Commercial reversed-phase cartridges were used for the separation of 3H2O which was produced in an aromatization reaction of androstenedione by human placenta in vitro. The assay is simple, rapid and reproducible. Metabolites originating from androstenedione were separated and quantified by thin layer chromatography. The microsomal fraction exhibited the highest aromatase activity which was inhibited (54%) by aminoglutethimide (500 microM) and by about 30% by alpha-naphthoflavone. Aromatase activity was not inhibited by known inhibitors of xenobiotic metabolism such as metyrapone or SKF 525A or by xenobiotic substrates such as 7-ethoxycoumarin or benzo(a)pyrene. Placental aromatase activity was not affected by maternal cigarette smoking. No correlation between aromatase and aryl hydrocarbon hydroxylase activities in the placentas from both smoking and non-smoking mothers was found. These results show that the aromatase activity in human placenta is catalysed by a distinct form of cytochrome P-450 which is different from forms with xenobiotic-metabolising activity, and also show that the aromatase activity is similar in placentas from both smoking and non-smoking mothers.