In view of the extensive use of estrogenic hormones by the human population, either as therapeutic agents, or in the composition of contraceptive pills, it is important to investigate more thoroughly the adverse biological effects of synthetic hormones. Diethylstilbestrol, ethynylestradiol, estradiol-17beta and estrone were chosen for our experiments. Evidence of carcinogenicity in rodents has been reported for each of these compounds, but so far only few studies have been carried out in vitro. Because it has been shown that isolated liver cells in suspension are able efficiently to metabolize steroid hormones, we have tested these chemicals in V79 cells with a cell-mediated system using primary hepatocytes from male and female rats as the metabolic layer. The incubation in the presence of the chemical to be tested was carried out at concentrations ranging from 25 to 100 microM, for 48 h before plating the V79 cells to score for mutagenicity or toxicity. In the absence of hepatocytes, the 4 estrogenic hormones were very toxic, but not mutagenic. The co-cultivation of V79 cells with primary hepatocytes decreased the toxic effect induced by the sex hormones, except in the case of ethynylestradiol. However, no mutation, determined as 8-azaguanine- or ouabain-resistance, was induced under these conditions by any of the hormones tested. The lack of mutagenic activity of these hormones in our assay had been confirmed by the use of primary liver cells that originated from a rat treated with Aroclor, an inducer of drug-metabolizing enzymes.