Parenchymal cells from adult rat liver, cultured in perifused monolayers, increased the levels of urea-cycle enzymes between 15% and 60% in response to glucagon within 24 h. This stimulation was drastically enhanced by the simultaneous presence of dexamethasone, especially in the case of argininosuccinate synthetase and argininosuccinate lyase, which increased nearly threefold. Dexamethasone itself produced only negligible stimulation, but exerted a similar effect on the stimulatory action of glucagon, if it was exclusively present during 6 h prior to the glucagon treatment, suggesting a permissive action of this hormone. The effect of glucagon, particularly in the presence of dexamethasone, was mimicked by dibutyryl adenosine 3':5'-monophosphate, whereas epinephrine was ineffective. All stimulations induced by hormones or dibutyryl adenosine 3':5'-monophosphate were abolished by cycloheximide, suggesting the involvement of protein synthesis in the induction process. Using the usual culture technique with a discontinuous supply of medium no significant effect of glucagon and dexamethasone could be measured. This striking difference between both culture systems indicates that perifusion is the more adequate in vitro system for studies of the regulation of enzyme levels. Possible reasons for the failure of hormonal stimulation of urea-cycle enzymes in normal monolayer culture are discussed.