³⁵S-ACaM2 was producted by using ³⁵S-labeled amino acid mixture in E.coli. SDS-PAGE and autoradiograph indicated that high-purified, high-specific radioactivity ³⁵S-ACaM2 was obtained.Electrophoresis character of ³⁵S-ACaM2 is the same as that of unlabeled ACaM2 with Ca²⁺ or EGTA. Dot-blot and NC overlay experiment showed that ³⁵S-ACaM2 could be used to detect calmodulin-binding proteins as a sensitive probe.