We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.