Rainbow trout hepatocytes were isolated by a two step perfusion technique and maintained either in monolayer culture for 5 days or in aggregate culture for 30 days. Cytochrome P450 content decreased from day 0 to day 5 in both culture systems, and then was preserved at the same level after one month in aggregated cells. 7-Ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyl transferase were not significantly different in freshly isolated cells and in 30-day aggregated hepatocytes, whereas a substantial increase in glutathione S-transferase was observed. Two-day exposure of cells to beta-naphthoflavone led to a significant increase in EROD activity in both culture systems, especially after one month of aggregation (10-fold increase). According to these results, aggregate culture of rainbow trout hepatocytes seems to be a promising in vitro model to investigate the biotransformation pathways in fish and their regulation by endogenous and exogenous compounds.