A strong lytic activity against Micrococcus lysodeikticus cell walls was detected in calf rennet. Purification was carried out using ion exchange, electrofocusing and gel-filtration. Hydrolysis of the bacterial peptidoglycan by the isolated enzyme released only reducing groups, thus indicating true glycolytic activity. Comparative tests with hen egg white lysozyme suggested a similar mode of action. - Multiple enzyme forms were revealed, with mol.wts of ca. 15,000, pH optima of ca. 5.0, and pl's ranging from 6.5 to 7.8. The enzyme exhibited remarkable stability against heat and pH conditions ranging from 2 to 9. Significant chitinase activity was observed at pH 5.0. - Specifie antisera revealed sharp precipitation arcs in the ß- to y-electrophoretic region, when diffused against the most acidic or basic molecular forms, respectively. - The rennet enzyme differs markedly from the bovine milk lysozyme described only by Chaudan et al. (BBA 110, 389, 1965).