Rats fed a purified L-amino acid diet with 0.72, 1.50, 2.9 or 4.3% lysine excreted 117, 124, 237 and 628 micrograms/day orotic acid, respectively. Dietary arginine supplementation prevented the orotic aciduria induced by excess dietary lysine. Increased orotic acid excretion was accompanied by a significant depression in urinary urea in rats fed a diet containing 4.3% lysine compared to those fed a diet containing 0.72% lysine. As measured by incorporation of [14C]HCO3, lysine addition to liver slices or isolated hepatocytes resulted in a progressive increase in the rate of orotic acid biosynthesis. The minimum quantity of lysine tested that significantly increased the rate of orotic acid biosynthesis was 0.5 mM or 1 mM for studies with slices and hepatocytes, respectively. Ammonia at concentrations of 0, 0.75, or 5.0 mM NH4Cl linearly increased orotate and urea synthesis. Inhibition of urea biosynthesis resulting from lysine supplementation coincided with an increase in pyrimidine biosynthesis. Addition of 1 mM arginine to the liver incubation media prevented the increased rate of orotic acid biosynthesis caused by lysine. Arginine addition may overcome an approximate 90% competitive inhibition of arginase by excess lysine.