The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca(2+)-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca(2+)-CaM complex were determined to be 2.1 +/- 0.1 and 2.0 +/- 0.2 microM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca(2+)-CaM, with dissociation constants of 1.8 +/- 0.2 and 2.8 +/- 0.9 microM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction.