De novo pyrimidine synthesis was studied in mouse liver, intestine, and kidney by intraperitoneal infusion of 15NH4Cl and analysis of 15N incorporation into uracil nucleotide pools. When the dose of a 1-h infusion of 15NH4Cl was increased from 50 mumol to 250 mumol the fraction of the total uracil nucleotide pool formed by de novo synthesis increased 4.0-fold in liver to 8.4% and 2.3-fold in intestine to 13.7%. The increase in intestine was independent of the increase in liver as evidenced by the lack of correlation between the increase observed in the intestine and liver of the same animal and the different distributions of label in the uracil ring nitrogens. A 2.4-fold increase in newly formed uracil nucleotides was observed in kidney when the infusion dose was raised from 150 mumol to 250 mumol. The increase in kidney was correlated with the increase in liver in the same animal and the distribution of label in the uracil ring nitrogens was similar to the distribution in liver. These results suggest that the increase in newly formed uracil nucleotides in intestine is due to increased de novo synthesis of pyrimidines in the intestine, while the increase in the kidney is due to increased salvage synthesis of uracil nucleotides from uridine synthesized in the liver and output to the circulation.