AbstractThe sperm of the toad,Bufo bufo japonicus, smeared on a gelatin film displays a “halo” reaction around the acrosomal region. The treatment of sperm with 0.5% Triton X‐100 yielded activity which lyses the vitelline coat (VC) of coelomic eggs, but not the fertilization coat of activated eggs. Electronmicroscopy showed that these detergent‐treated sperm had lost the plasma membrane and the acrosomal cap, indicating acrosomal localization of the VC lysin. The VC lysin was able to lyse the VCs of several heterologous anuran species, and this activity correlated well with the occurrence of cross‐fertilization byBufosperm. Proteolytic activities of the VC lysin were quantitated employing N,N‐dimethylated VC proteins or casein as substrates. Successive fractionation by NH42SO4precipitation, DEAE‐cellulose chromatography, and Sephadex gel filtration yielded a 100‐fold increase of proteolytic activity compared with that of the original detergent extract. On gel filtration, the VC lysin activity was recovered both in the M.W. 54,000 fractions and the considerably higher (>100,000) M. W. fractions. Part of the latter activity was recoverable in the former after the treatment with urea and rechromatography. The VC lysin was most active at pH 7.4, but its activity was lost by preincubation at temperatures higher than 60°C. The activity was inhibited considerably by serine‐protease and chymotrypsin inhibitors, but not at all by trypsin inhibitors. The lysin was unable to hydrolyze arginine or tyrosine esters, but was able to hydrolyze active site titrants for chymotrypsin more effectively than for trypsin. In all aspects the VC lysin, although sharing some properties with serine‐proteases such as chymotrypsin, is a unique proteinase(s) well adapted to its r