Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.