5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. However, the role of ribose-5-phosphate (R5P), the precursor for PRPP, in the regulation of PRPP and de novo purine synthesis has not yet been clarified conclusively. This study was designed to clarify interrelationships between R5P content, PRPP availability, and the rate of de novo purine synthesis in the cultured human hepatoma cell line (HepG2 ), a plausible model for normal human hepatocytes. Increasing glucose concentration in the culture media from 0 to 10 mmol/L resulted in a 2.9-fold elevation of cellular R5P content (from 107 ± 31 to 311 ± 57 nmol/g protein), associated with a correlated increase of 7.14-fold in cellular PRPP availability (from 4.76 ± 3.4 to 34 ± 8.4 pmol/mg protein/min) and of 149-fold in the rate of de novo purine synthesis (from 55 to 8,204 dpm/mg protein/h). Plotting the rate of de novo purine synthesis versus R5P content indicates that at a wide range of R5P content, including that prevailing in hepatocytes under physiological conditions, the rate of purine synthesis depends on R5P content. A similar dependence was also demonstrated for PRPP availability. The rate of de novo purine synthesis exhibited a sigmoidal dependence on PRPP availability. The demonstration in human hepatocytes of dependence of the rate of purine synthesis on R5P content has implications concerning the pathogenesis of purine overproduction associated with several inborn and acquired conditions in man.