Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 μM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca² uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca² influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.