I n previous studies from this laboratory, human bone marrow hypoxanthine concentrations were found to average 7.1 microM, three times higher than plasma hypoxanthine concentrations measured simultaneously. To assess the significance of this finding, the relationship between hypoxanthine concentration and the rate of purine nucleotide synthesis by the de novo pathway was studied in normal human bone marrow mononuclear cells and in the human promyelocytic cell line, HL-60, in vitro. Utilizing a [14C]formate incorporation technique, rates of total cellular de novo purine synthesis as well as rates of de novo adenine, de novo guanine, and thymine synthesis and incorporation into RNA and DNA were measured as a function of hypoxanthine concentration. In normal human marrow cells, the rate of total de novo purine synthesis declined by 81%, while the rate of de novo adenine and de novo guanine synthesis and incorporation into macromolecules declined by 89 and 75%, respectively, when media hypoxanthine was increased from 0 to 10 microM. Similar results were seen in the HL-60 cell line. In contrast, rates of thymine synthesis and incorporation into DNA as well as overall rates of RNA and DNA synthesis did not change with varying media hypoxanthine concentrations. In addition, hypoxanthine salvage and incorporation into RNA and DNA was shown to progressively increase with increasing media hypoxanthine concentrations. These results indicate that physiologic concentrations of hypoxanthine are sufficient to regulate the rate of de novo purine synthesis in human bone marrow in vivo.