The Ca2+-dependent conformational alteration of the brain-specific S-100 protein was studied by reacting the protein with N-ethyl[2,3-14C]maleimide in the absence and presence of Ca2+ and under denaturing conditions. Peptic hydrolysates of the 14C-labeled protein were analyzed and fractionated by high-performance liquid chromatography. Labeled peptide fractions were characterized by high-voltage electrophoresis and TLC. A clear distinction could be made between two classes of sulfhydryl-containing fragments: (a) neutral, hydrophobic, and (b) acidic. Ca2+ markedly favored 14C incorporation into the former components, whereas the latter were readily available only under denaturing conditions.