Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene (AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced microsomes and with six highly purified cytochrome P-450 isoenzymes from rabbit liver. Kinetic parameters were defined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylation was best defined by a two enzyme system, displaying a high affinity and relatively low capacity and a low affinity high capacity components in both control and TCDD induced microsomes. All the purified cytochrome P-450 isoenzymes were capable of catalyzing the 7-hydroxylation of AAF and, with the exception of form 4, this was the only oxidation on the AAF molecule catalyzed by these forms. It is probable that forms 1, 4 and 6 accounted for a substantial part (greater than or equal to 25%) of total metabolic capacity corresponding to the high affinity component of 7-hydroxylation, whereas forms 3b and 3c accounted for less than 5% of the metabolic capacity displayed by the low affinity component in control microsomes. However, forms 4 and 6 could account for greater than 90% of the metabolic capacity of the high affinity component of 7-hydroxylation in TCDD microsomes, whereas the form(s) responsible for the metabolic capacity of the low affinity component were not identified. Each of the 1-, 3-, 5- and N-hydroxylations were best defined by a single enzyme system in both control and TCDD microsomes (3- and 5-hydroxylations could not be defined in TCDD microsomes). Close agreements were found between the apparent Km for N-hydroxylation in control, TCDD induced microsomes and with form 4. alpha-Naphthoflavone inhibited AAF N-hydroxylation to a similar extent in control and TCDD microsomes and in form 4. These date indicate that: a subpopulation of cytochrome P-450 isoenzymes, which includes all the purified P-450 forms tested in the present study, is solely involved in detoxification (i.e., 7-hydroxylation) of AAF, and as such probably behave as a functional unit in vivo; modulation of cytochrome P-450 content by inducers such as TCDD results in emergence of relatively few cytochrome P-450 isoenzymes that can account for most of the oxidative metabolism of AAF; and a single cytochrome P-450 isoenzyme (i.e., form 4) is responsible for catalyzing N-hydroxylation of AAF, the first and the obligatory step in the metabolic activation of this carcinogen.