Effects of five organic solvent vehicles on benzo(a)pyrene- hydroxylase (BP-hydroxylase) activity and on the benzo(a)pyrene (50328) (BP) metabolite profile were studied in lung microsomes prepared from male New Zealand white rabbits. The production of 3- OH-benzo(a)pyrene (13345216) and 9-OH-benzo(a)pyrene (17573216) was used to evaluate the effects of dimethyl-sulfoxide (DMSO), acetone, methanol, ethanol, and ethyl-acetate on BP-hydroxylase activity. A final concentration of 2.5 milligrams/milliliter (mg/ml) of BP was delivered to microsomal reaction mixtures in solvent volumes ranging from 10 to 50 microliters (microl). The amounts of 3-OH- benzo(a)pyrene and 9-OH-benzo(a)pyrene were determined by measuring fluorescence with a Turner spectrofluorometer. High performance liquid chromatography was used to obtain the BP metabolite profile from microsomal mixtures with final BP concentrations of 0.5mg/ml solvent and 2.5mg/ml solvent. All five vehicles exhibited dose dependent inhibition on the activity of BP-hydroxylase. When BP was delivered in 10microliters of each solvent, enzyme activities were greatest in DMSO followed by methanol, ethanol, acetone, and ethyl- acetate. Increasing solvent volume produced dose dependent inhibition in BP-hydroxylase activity in each case. At 50microl of solvent volume, BP-hydroxylase activities were greatest in DMSO followed by methanol, acetone, ethanol and ethyl-acetate. HPLC analysis showed that the production of metabolites was greatest and the linearity of product formation retained longest when DMSO was used. The particular solvent used affected the type, magnitude, and time course of all BP metabolites. The authors suggest that DMSO is probably the best solvent vehicle for studying BP metabolism in rabbit lung microsomes.