By using various humectant systems, the specificity of hydrolysis of _s1-, -, and -caseins by the cell envelope-associated proteinase (lactocepin; EC 3.4.21.96) with type P₁ specificity (i.e., lactocepin I) from Lactococcus lactis subsp. lactis BN1 was investigated at water activities (a_w) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (a_w = 0.95), hydrolysis of -casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type P_III enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from -casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (a_w = 0.99). In addition, _s1-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an a_w of 0.95) was generally as expected for lactocepin I activity except that -casein peptide 47-52 and -casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of a_w and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.