Thiosubstrates in which the thio group is located at or near the vicinity of hydroxylation may be used as biochemical probes to correlate the amount of cytochrome P-450 associated with specific hydroxylases. Incubation of deacetylspironolactone (17 beta-hydroxy-7a-mercapto-3-oxo-pregn-4-ene-21-carboxylic acid gamma-lactone) with guinea pig adrenal microsomal suspensions and NADPH causes a 40 to 50% loss of cytochrome P-450 and a 70-80% loss in the activity of 17 alpha-hydroxylase but no decrease in 21-hydroxylase activity. In vitro studies with 7 alpha-thiotestosterone have shown that a 5-10% loss of hepatic cytochrome P-450 resulted in a 30 to 40% loss in the activity of testosterone 7 alpha-hydroxylase but not of testosterone 2 beta, 6 beta, and 16 alpha-hydroxylase. Similar in vitro studies with 7-thiobenzo(a)pyrene have indicated that a loss of hepatic cytochrome P-450 is associated with a preferential decrease in the rate of benzo(a)pyrene oxygenation at the C-7 position but not at carbon positions 1,3,5, or 9.