Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.