Mechanisms leading to the formation of crystalline inclusions in the cisternal space of the rough endoplasmic reticulum are poorly understood. This phenomenon was investigated in pancreatic acinar cells using two different experimental models: 1) Intraperitoneal injection of DL-p-chlorophenylalanine methyl ester, and 2) culture of isolated acinar cells within the Matrigel basement membrane in the presence of 2% dimethyl sulfoxide. Features and composition of induced crystals were analyzed by protein A-gold and lectin-gold cytochemistry, electron microscope autoradiography, electron energy loss spectroscopic imaging and energy dispersive X-ray analysis. Crystal formation occurred in ribosome partially free rough endoplasmic reticulum (RER) regions and was similar in both experimental protocols. The protein A-gold revealed the presence of nine major pancreatic enzymes in the crystals. However, the labeling intensities varied among enzymes with higher concentrations of amylase than chymotrypsinogen when compared to the secretory granules. Concanavalin A and Helix pomatia labelings were weak over the crystals and did not correspond to those of RER or secretory granules. Sulfur contents in crystals were lower than phosphorus and their ratio was opposite to the one found in secretory granules. Electron microscope autoradiography demonstrated incorporation of radiolabeled leucine and presence of newly synthesized proteins in the crystals. Furthermore, cells containing both crystals and secretory granules displayed silver grains in most of the cellular compartments involved in secretion. Thus, failure in the normal concentration and sorting process of secretory proteins leading to crystal formation includes changes in protein glycosylation and decrease of disulfide bond formation while retaining secretory capabilities.