The regulation of flavin-containing monooxygenase (FMO) by thyroid hormones was examined under well defined in vitro conditions using adult male rat hepatocytes co-cultured with rat liver epithelial cells of primitive biliary origin. Serum free medium was used to avoid interferences from foetal bovine serum. The effect of thyroxine (T4) and its major metabolite l-triiodothyronine (T3) on FMO activity was estimated spectrophotometrically by measuring the rate of methimazole oxygenation. The highest non-cytotoxic doses of T3 and T4 that could be used in co-cultures were determined by measuring both lactate dehydrogenase leakage into the medium and microsomal protein content of the hepatocytes as a function of culture time. In addition, hormonal responsiveness of the in vitro system used was confirmed by malic enzyme activity measurements. Administration of 10 mum T3 or T4 was found to cause a significant decrease in FMO activity and content, suggesting a suppressive role of both hormones on the regulation of FMO activity in male rats.