The relationship between the metabolism and hepatotoxicity of hydrazine (Hz) was studied in an isolated rat hepatocyte system. After incubation at 37°C for 60 min; the concentration of Hz decreased to 34% of the initial value. (from 98 to 33 nmol/ml). A further pronounced loss was observed when Hz was incubated with hepatocytes isolated from rats which had been pretreated with phenobarbital (PB) or rifampicin. Since metyrapone and piperonylbutoxide inhibited the metabolism in the systems obtained from both PB-pretreated rats and untreated rats, an important role of hepatic cytochrome P-450 in Hz oxidation was suggested. The difference spectrum of Hz with cytochrome P-450 in isolated rat hepatocytes demonstrated the formation of complexes between metabolic intermediates of Hz and cytochrome P-450. The trypan blue exclusion test, the measurement of K⁺ level and the determination of glutathione content retained in the cells were also performed. The results suggested that Hz is cytotoxic to isolated rat hepatocytes.