The metabolism of aflatoxin B1 (AFB1) was examined in freshly isolated hepatocytes from rainbow trout. Intracellular DNA adduct formation was linearly related to AFB1 dose, and qualitatively similar to adducts formed in vivo. The rate of adduct accumulation was constant during the first hour following completion of the preparation, after which an increase and gradual decrease in rate routinely occurred. The relative rates of production of the major unbound AFB1 metabolites aflatoxicol, aflatoxin M1, and polar conjugates, also remained constant over the first hour of preparation age, but subsequently changed in a manner consistent with the changes in DNA binding. The common solvent vehicles ethanol and dimethyl sulfoxide were shown to seriously perturb AFB1 metabolism and DNA binding even at levels less than 1%. A simple method is reported for removal of ethanol prior to introduction of hepatocytes for incubation with AFB1. The influence of cell concentration was also examined. DNA binding and relative distribution of AFB1 metabolites showed little or no dependence in the range 1-6 x 10(6) cell/ml, but were substantially altered above 10(7) cells/ml. Under defined conditions, studies in isolated hepatocytes appear to reflect in vivo cell capabilities for AFB1 metabolism.