Cultured rat hepatocytes were exposed to hydrazine for 4 hr, 17 hr or 4 hr followed by a 13-hr post-exposure period. Hydrazine was cytotoxic as measured by leakage of lactate dehydrogenase (LDH), caused depletion of ATP and inhibited protein synthesis. The cytotoxicity and depletion of ATP in cultures exposed to hydrazine for 4 hr was less than that previously reported in hepatocyte suspensions exposed for 4 hr (Ghatineh et al., Archives of Toxicology 1992, 66, 660). The threshold cytotoxic concentration (20 mm) was also higher in cells in culture than in cells in suspension (16 mm). Inhibition of protein synthesis was detected at a much lower concentration of hydrazine (0.5 mm) than was required to deplete ATP (16 mm) or cause cytotoxicity (20 mm). ATP depletion and inhibition of protein synthesis were similar after a 4-hr exposure with or without a 13-hr post-exposure period, but leakage of LDH still occurred during this period. After the 17-hr exposure, the leakage of LDH, ATP depletion and inhibition of protein synthesis were greater and the threshold concentration of hydrazine required for a significant effect on all three parameters was lower. This was so whether compared with a 4-hr exposure, or a 4-hr exposure plus a 13-hr post-exposure period. The results of this study indicate the following: (a) the sensitivity of cultured hepatocytes to hydrazine is no greater than that of hepatocytes in suspension; (b) the duration of exposure to hydrazine is important but the effect depends on the parameter measured; (c) hydrazine causes a dose-dependent inhibition of protein synthesis at much lower concentrations than those causing LDH leakage; (d) maintenance of cytochrome P-450 in cultured hepatocytes by exposure to metyrapone did not alter the cytotoxicity of hydrazine.