Urea cycle enzymes of rats were altered by feeding protein-free 15% casein, or 70% casein diets. Cells isolated from the livers of these rats were incubated with 10 mM ammonia and varying levels of ornithine (2 or 10 mM) and lactate (0-10 mM). Conditions providing the greatest rate of urea synthesis depended on the diet consumed and on whether rats were fed or starved 24 hours before cell isolation. Maximum rates of urea synthesis, though not different for the fed and starved groups, increased with the protein content of the diet and were equal to the maximum theoretical rates, as determined by argininosuccinate synthetase activity. This indicates that with optimal conditions, the activity of argininosuccinate synthetase is the rate-limiting factor in the synthesis of urea in isolated hepatocytes.