The cytotoxic response to aflatoxin B(1) (AFB) was investigated in RTL-W1, a cell line derived from the normal liver of a mature rainbow trout (Oncorhynchus mykiss). AFB altered RTL-W1 morphology and ultrastructure and inhibited DNA synthesis. The effective concentration required for 50% inhibition (EC(50)) of DNA synthesis, after 2 days of treatment was 0.04 mug/ml. This response was compared with that of two other salmonid, but non-liver, cell lines [rainbow trout gonad (RTG-2) and Chinook salmon embryo (CHSE-214)]. Although RTG-2 cells were as sensitive as RTL-W1 cells (EC(50) for inhibition of DNA synthesis was 0.05 mug/ml), CHSE-214 cells were unresponsive to AFB at concentrations as high as 2 mug/ml. After a single AFB exposure, RTL-W1 sublines were isolated that had phenotypic changes typical of malignant transformation. These were increased growth rate, reduced contact inhibition of growth, altered cellular morphology and growth in soft agar. In addition, RTL-W1 metabolized AFB: the major metabolites, aflatoxin (AFL) and aflatoxin M(1) (AFM), were detected by thin-layer chromatography and HPLC. The relative amounts of these metabolites, unlike those observed with RTG-2 cells, were in close agreement with those produced by trout liver in vivo. Thus, RTL-W1 could provide a sensitive in vitro model system for studying the action of biotransformation requiring xenobiotics. Overall, the observed responses were similar to those reported for liver cells in AFB-exposed trout, suggesting that RTL-W1 cells are suitable for studying cytotoxic effects and malignant transformation in vitro.