The effects of purine nucleosides on 5-phosphoribosylpyrophosphate (PP-ribose-P) synthesis were investigated in human erythrocytes in vitro. Previous observations have shown an increase of PP-ribose-P formation with nucleoside when inorganic phosphate (Pi) exceeded 20 mM. In contrast, when external Pi varied from 0 to 25 mM adenosine, inosine, guanosine, or 6-methylmercaptopurine riboside 1.25 mM decreased intracellular PP-ribose-P. The mechanism of this nucleoside effect on P-ribose-P concentration was investigated. Decreased synthesis rather than increased utilization accounted for the nucleoside effect. A decrease of erythrocyte Pi from the control values of 0.5 to 2.4 mM accompanied the nucleoside-related diminution of PP-ribose-P synthesis. This reduction of Pi was capable of decreasing the activity of PP-ribose-P synthetase which is known to be sensitive to small changes of Pi. The decrease of erythrocyte Pi levels was caused both by those nucleosides which were eventually degraded via purine nucleoside phosphorylase (adenosine, inosine, and guanosine) and by those nucleosides which were substantially phosphorylated initially by adenosine kinase (methylmercaptopurine riboside and adenosine with erythro-9(2-hydroxyl-3-nonyl)-adenine). Twenty-five per cent of methylmercaptopurine riboside was converted to the monophosphate derivative during the incubation. The presence of this compound, capable of inhibiting PP-ribose-P synthetase, provided evidence for a second mechanism of inhibition of PP-ribose-P synthesis by methylmercaptopurine riboside. No substantial increase of AMP, ADP, 2,3-diphosphoglycerate, or cyclic AMP nor a decrease of ribose 5-phosphate was found. These observations suggest that under physiological Pi concentrations nucleosides diminish PP-ribose-P synthesis mainly by decreasing intracellular Pi. Regulation of PP-ribose-P formation by alterations of intracellular Pi levels may be an important control mechanism and may account for a number of biological effects of nucleosides.