The apparent pH dependence and activity of subtilisin dispersed in organic solvents have been studied. The data demonstrated that the apparent pH dependence displayed by the enzyme in organic media resembled that in water in a broad sense, but it was dependent upon solvent type, water content, and method of water delivery. The effect of organic solvents on the apparent pH dependence of suspended subtilisin powder can be related to the change in polarity of the enzyme’s active site. The apparent pK(a) of subtilisin was more affected by changing water content than by changing solvents, and it was significantly lower when water was delivered by sodium phosphate salt hydrate (Na4P2O7.10H2O) than when 0.05% (v/v) water was added directly. The activity of the enzyme in organic media did not correlate to the classical scales of solvent hydrophobicity.