Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. Km values for activity showed a difference between the cellular fractions with a Km of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The Vmax values observed were 0.83 nmol min− (nmol microsomal P-450) −1 and 0.4 nmol min−1 (nmol cytosolic P-450)−1. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.