The importance of oxygen in maintaining the functional integrity of hepatocytes has been well established in a variety of experimental models, such as in vivo, perfused liver and isolated hepatocytes. However, one of the shortcomings of these systems is their short life span. Therefore, we have examined the effects of long-term hypoxia on cellular adenine nucleotide content and cellular functions, such as albumin production, urea production and DNA synthesis, in adult rat hepatocytes in primary culture. Hepatocytes were cultured at a density of 11 x 10(4) and 5 x 10(4) cells/0.18 mL per cm2 for the study of albumin and urea production and DNA synthesis, respectively, at various oxygen tensions (20, 12, 8 and 5%) for 24 h. Cellular ATP content in cultured hepatocytes in hypoxia gradually declined, corresponding to the decrease in oxygen tension, and the cellular ATP level at 5% oxygen was approximately 20% of that at 20% oxygen. Albumin production also decreased in parallel with the decrease in cellular ATP content in cultured hepatocytes in hypoxia. However, even when cellular ATP content gradually declined corresponding with the decrease in oxygen tension in cultured hepatocytes in hypoxia, such as at 8 or 5% oxygen, urea production remained at a high level; in contrast, DNA synthesis was completely suppressed. These results suggest that the cellular ATP content decreases in cultured hepatocytes during long-term hypoxia in relation to oxygen tension and that the relationship between decreased ATP levels and liver function in cultured hepatocytes during hypoxia differs for albumin production, urea production and DNA synthesis.