Sensitive and specific chromatographic assays for the measurement of flavin-containing monooxygenase (N-oxygenase and S-oxygenase activities) and aldehyde oxidase activities in rat hepatocyte primary cultures were developed. Conditions for the measurement of enzymatic activities in rat liver cell cultures were first optimized using freshly isolated cell suspensions. Activities of the cytochrome P-450/P-448 isozymes in rat hepatocytes maintained in primary culture [assessed by the O-deethylation of 7-ethoxycoumarin (P-450/P-448) and the N-demethylation of N,N-dimethylaniline (P-450)] rapidly declined to 25% of the initial levels by 48 hr in culture. The flavin-containing monooxygenase system was considerably more stable in cell culture. Flavin N-oxygenase activity (assessed by the N-oxidation of N,N-dimethylaniline) declined slightly (10-15%) and remained almost constant over the 48-hr culture period, whereas S-oxygenase activity (assessed by the S-oxygenation of tetrahydrothiophen) gradually declined and stabilized at approximately 65% of its initial activity at 48 hr in culture. Aldehyde oxidase activity (assessed by the 1-hydroxylation of phthalazine) declined to approximately 20% of the initial value by 48 hr in culture. The differential stability of the microsomal and cytosolic drug oxidases in rat hepatocytes in primary culture demonstrates some of the limitations of this model for metabolic studies.