Until now hormonal regulation of flavin-containing monooxygenase (FMO) has been studied to a limited extent and all experiments reported so far have been carried out in vivo using invasive techniques. In order to examine whether in vitro models could be applied to study the regulation of FMO, pure cultures of rat hepatocytes and their co-cultures with primitive bile-duct epithelial cells have been used. Since the addition of foetal calf serum (FCS) to the medium, usually done to improve cell attachment, could interact with some hormones, the effect of its removal on FMO activity has been investigated. Furthermore, to find out if in vitro results comparable with the in vivo situation could be obtained, phenobarbital was added and its effect on the FMO system was measured. In pure hepatocyte cultures FMO activity declined continuously as a function of culture time, and after 6 days it was only 15% of that obtained for freshly isolated hepatocytes. In contrast, in co-cultures after an initial decrease a steady-state situation was maintained at a level of approximately 37% of the initial value. This observation was noted from day 7 onwards and for at least 1 wk. It was also observed that in both culture systems, the various media had no statistically significant effect on FMO activity expressed in nmol methimazole/min/mg microsomal protein. In pure hepatocyte cultures, however, addition of FCS and phenobarbital significantly increased the microsomal protein content per culture dish in comparison with the value obtained for serum-free medium. In the latter case deterioration and loss of hepatocytes in the absence of FCS occurred. In conclusion, the expression of FMO is better and longer maintained in co-cultures than in pure cultures. The removal of FCS from the medium in co-cultures does not affect FMO activity. Since addition of phenobarbital has no inducing effects on FMO activity, as is also the case in vivo, the results support the idea of using co-cultures of rat hepatocytes as an in vitro model for regulation studies of FMO.