The influence of microsomal concentration on unbound fraction (fu(mic)), half-life (t(1/2)), apparent intrinsic clearance (CL(int,app)) and apparent Michaelis-Menten constant (K(m,app)) was examined for two compounds, one representative of high nonspecific binding to microsomes (compound A) and one representative of low (compound B). Kinetic parameters were estimated for the two probe compounds at two human microsomal protein concentrations (0.46 and 2.3 mg/ml) and cytochrome P450 concentrations (0.20 and 1.0 microM), representing a 5-fold difference in microsomal concentration. For compound A, fu(mic) and CL(int,app) were inversely proportional to microsomal concentration. Conversely, the K(m,app) of compound A was proportional to microsomal concentration and the half-life was unchanged. For compound B, half-life was inversely proportional to microsomal concentration. In this case, fu(mic), CL(int,app), and K(m,app) were not proportionally influenced. The experimental observations were entirely consistent with that predicted by a mathematical relationship between microsomal concentration, fu(mic), t(1/2), CL(int,app), and K(m,app). These results demonstrate that when nonspecific binding is extensive, CL(int,app) is dependent on the arbitrary choice of microsomal concentration included in the incubation.