The objective of this study was to investigate the characteristics and distributions of neuronal origin of cerebellar afferents from motor cranial nerve nuclei innervating extraocular muscles by the method of retrograde transport of two fluorescence tracers in rats. Under deep anesthesia and aseptic conditions, 5 microl of 3% solution of Fluoro-Gold (FG) in phosphate buffer solution (PBS) was injected into the bellies of the six extraocular muscles to study the labeling of motoneurons innervating corresponding extraocular muscles. The cerebellum was exposed by craniotomy, and 0.3 microl of 10% solution of Dextran Tetramethyl Rhodamine Biotin (Micro Ruby: or MR) in PBS was injected into many regions of the anterior vermis (lobule I, II) and the posterior vermis (lobule VI, VII, IX, X), the flocculus, the paraflocculus and the deep cerebellar nuclei. Multiple injections were made to cover the entire cerebellum in order to obtain a near maximum labeling of cerebellar afferent neurons. In other cases, only small single or a few injections were made in specific areas of the cerebellum to study specific distributions and topographic organization. In one group of rats, injections were made both in the extraocular muscles with FG and in the cerebellum with MR to study the double labeling of neurons, which project their axons to both the extraocular muscle and the cerebellum. Another group of rats were injected in both sites with only PBS and served as the control for auto-fluorescence background. After 3 days postoperative survival time, all animals were deeply reanesthetized and perfused with heparinized normal saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and 30% sucrose solution in PBS. The brainstem and the cerebellum were removed immediately, and stored in sucrose solution in PBS at 4 degrees C. Serial transverse sections of the brainstem and sagittal sections of the cerebellum were obtained by a freezing microtome at 40 microm thickness, collected on uncoated glass slides, and immediately dried. All sections were examined under an epifluorescence or confocal microscope equipped with filter systems for FG and MR. The presence of both single and double retrograde labeled neurons in the Oculomotor (CN 3), Trochlear (CN 4) and Abducens (CN 6) nuclei was recorded, photographed, stored as computer images files and printed out as hard copies. The labeling neurons in the vicinity of the CN 3, 4, 6 from all sections were plotted onto diagrams and counted Neurons labeled only with MR retrogradely transported from injection sites in the cerebellum were found bilaterally and scattered throughout in the Oculomotor, Trochlear and Abducens nuclei. These neurons labeled only with MR were small and medium-sized interneurons and represented only a small proportion of the entire population. Neurons labeled only with FG retrogradely transported from injection sites in the extraocular muscles were the most numerous, and distributed almost throughout the entire population of small, medium-sized and large motoneurons, which innervate the extraocular muscles. A smaller proportion of small and medium-sized FG labeled neurons within these nuclei were also double labeled with MR, indicating that they project their axon collaterals to both extraocular muscles and the cerebellum. In conclusion, the present findings provide clear anatomical evidence that a small population of motoneurons in the Oculomotor, Trochlear and Abducens nuclei of the rat project their axon collaterals directly to the cerebellum and the extraocular muscle, in addition to the cerebellar afferents from other interneurons within these nuclei. The findings also indicate that cerebellar neuronal circuits play more direct roles in monitoring and controlling eye movements than previously known.