Methods were developed for the isolation and primary culture of rainbow trout (Salmo gairdneri) and channel catfish (Ictalurus punctatus) liver cells. Using a two-step perfusion technique, I isolated an average of 2.75 and 2.87 liver cells/g body weight from the trout and catfish, respectively. Hepatocytes represented 91.4% (trout) and 90.1% (catfish) of the total liver cells isolated. Both catfish and trout hepatocytes in primary culture displayed a linear decrease in survival with increased duration of culture. The DNA synthesis in the hepatocytes during culture showed a similar decrease with increased time in culture. Approximately 2.8% (trout) and 3.5% (catfish) of the hepatocytes exhibited nuclear labeling with [3H]dThd immediately after isolation. The labeling index decreased for hepatocytes from both species to 0% by the tenth day of culture. Activities of cytochrome P-450 and B-5 initially declined rapidly for both trout and catfish hepatocytes after placement in culture; however, these activities leveled off at low but measurable values for the first 8 days of culture. Unscheduled DNA synthesis (UDS) was induced in catfish and trout hepatocytes after exposure to dimethylnitrosamine, aflatoxin B1, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine. Trout hepatocytes displayed a decrease in UDS induction with aflatoxin B1 with increased age of the cultures. However, UDS induced by N-methyl-N'-nitro-N-nitrosoguanidine remained constant throughout the culture period.