Preincubation of guinea pig hepatic, pulmonary, or adrenal microsomes with cadmium or zinc decreased mono-oxygenase [benzo(a)pyrene hydroxylase, benzphetamine demethylase] activities. Addition of the same concentrations of the metals to the microsomal suspensions after the preincubation period had little or no effect on enzyme activities. The decline in mono-oxygenase activities produced by cadmium or zinc was dependent on the length of the preincubation period as well as the concentration of metal present during the preincubation. In addition, the preincubation effects of both metals were temperature dependent; at temperatures between 4 and 37 degrees C, loss of enzyme activity increased with increasing temperature. Cadmium and zinc produced greater decreases in mono-oxygenase activities in pulmonary and adrenal microsomes than in hepatic microsomes. The results indicate that the duration of exposure of hepatic and extrahepatic microsomal preparations to cadmium or zinc in vitro is an important determinant of effects on mono-oxygenases.