To identify the ovarian cell type(s) responsible for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and their dependence on hormonal influences, DMBA mono-oxygenase activity was determined in primary cultures of cells dispersed from rat ovaries and separated by centrifugation on discontinuous Percoll density gradients. The contents of progesterone and oestradiol in the different cell cultures were characterized. Moreover, the morphological appearance of ovarian cells obtained from untreated and pregnant mare's serum gonadotropin (PMSG)-treated rats was examined. It is concluded from experiments with immature PMSG-treated rats, that DMBA mono-oxygenase activity is localized in follicular granulosa (and/or theca) cells. This activity decreases during luteinization, and is recovered in a population of cells harvested at a higher density on the Percoll gradient. The xenobiotic-metabolizing activity was not correlated to the rate of biosynthesis of progesterone or oestradiol in isolated cells, measured in the presence or absence of human chorionic gonadotropin and/or testosterone. However, a certain dependence of DMBA metabolism on steroids and/or steroid-synthesizing enzymes could not be excluded. For example, DMBA mono-oxygenase activity was markedly increased in a cell population, tentatively identified as granulosa cells, obtained from untreated mature rat ovaries upon addition of testosterone, which is the substrate for oestrogen synthesis, to the cell culture.