The specific inhibitor of cysteine proteinases, cystatin C, was purified from ram rete testis fluid and the conditioned medium of Sertoli cells. This molecule associated with sheep liver cathepsin L at one of the fastest rates ever described for a proteinasehnhibitor interaction (1.75 ?0.20X108 M-' . s-'). But the association rate constant for the interaction of cathepsin L with a,-macroglobulin, a nonspecific inhibitor of proteinases, was also extremely high (8.8-C0.75X106 M-' . s-I). Cathepsin L complexed with a,-macroglobulin was protected from inhibition by type 2 and type 3 cystatins. The data indicate that cystatin C is the most potent inhibitor of cathepsin L in mammalian male genital tract fluids, whereas a,-macroglobulin may act as a terminal acceptor of this enzyme. These inhibitors could therefore inhibit the activated form of procathepsin L which may appear during the complex process of spermatozoa production and maturation in the testis and epididymis.