The endogenous biosynthesis of nitrate in rats was investigated by using 15NH3 administered as a continuous intravenous infusion for as long as 96 h. A comparison of the enrichment of 15N in urinary nitrate after a 24 h infusion revealed that it was 36% of the enrichment of plasma NH3 and about 50% of the enrichment of plasma urea and urinary NH3. Continuous infusion of 15NH3 for 96 h showed that a plateau for the incorporation of NH3 into nitrate is reached by 24 h, whereas the enrichment of urinary NH3 and urea increase during the 96 h. After the infusion of progressively larger doses of 15NH3, the concentration of nitrate synthesized de novo increased. Although there was a significant correlation between plasma 15NH3 concentration and 15NO3- appearance, a given change in plasma NH3 concentration does not produce a direct proportional change in nitrate synthesis. Our findings indicate that NH3 is a quantitatively significant nitrogen precursor for nitrate, but that approx. 50% of nitrate nitrogen is derived from other, as yet unidentified, sources.