To study the role of purine ribonucleotides as possible regulators of the rate of de novo purine biosynthesis in living human cells, we measured intracellular ribonucleotide concentrations by high-pressure liquid chromatography in a series of cloned human lymphoblast mutants selected by resistance to 8-azaguanine, in which the severity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency could be correlated with increases in the rate of de novo purine biosynthesis and increases in intracellular concentrations of phosphoribosyl pyrophosphate (PP-ribose-P). Compared with appropriate normal controls, intracellular purine ribonucleotide concentrations were not reduced in HGPRT-deficient lymphoblasts but there were striking increases in intracellular concentrations of some pyrimidine nucleotides and nucleotide sugars which appeared to be related to the degree of the deficiency. Similar changes were found in lymphoblasts from a Lesch-Nyhan boy. These data support the hypothesis that the accelerated rate of purine biosynthesis in HGPRT-deficient cells result from increases in intracellular PP-ribose-P concentration and not from changes in intracellular purine ribonucleotide concentrations. The possibility that the abnormality of pyrimidine nucleotide metabolism results from coordinate regulation of purine and pyrimidine biosynthesis by PP-ribose-P was not substantiated by measurement of rates of pyrimidine synthesis and experimental elevation of intracellular concentrations of PP-ribose-P after incubation of cells with inorganic phosphate.