The first steps of proteolysis of bovine beta-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it has been possible to selectively study the hydrolysis of two bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where beta-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192-193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189-190 was negligible. The following results were obtained; k1cat = 1.54 s-1, K1m = 0.075 mM, and k1cat/K1m = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192-193 by integrating the Michaelis equation: k2cat = 0.056 s-1, K2m = 0.007 mM, and k2cat/K2m = 79.7 mM-1 s-1. Under the latter conditions the four enzymic reactions involved in the cleavage of bonds 189-190 and 192-193 were first-order reactions. The four corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of four molecular species measured during the reaction period and those calculated by using the four apparent rate constants.