The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H2O2) and benzo[a]pyrene (B[a]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H2O2 for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[a]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 mu M after 4 hr of exposure. After 48 hr of exposure to B[a]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.