Pyrroline-5-carboxylate, the intermediate in the interconversion of proline, ornithine, and glutamate, increases 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) and purine nucleotide formation in intact human erythrocytes. We proposed that: 1) pyrroline-5-carboxylate is converted to proline by pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH, 2) NADP+ augments glucose-6-phosphate dehydrogenase activity, and 3) production of ribose-5-phosphate via the pentose shunt is increased. Since glucose-6-phosphate dehydrogenase plays a central role in this proposed mechanism, we examined the responsiveness of glucose-6-phosphate dehydrogenase-deficient erythrocytes to pyrroline-5-carboxylate. We compared erythrocytes from four Sardinian glucose-6-phosphate dehydrogenase-deficient subjects and four Sardinian normal controls. Without pyrroline-5-carboxylate treatment, the levels of pentose shunt activity, PP-ribose-P, and inosine monophosphate were comparable in the two populations. However, the response to pyrroline-5-carboxylate in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects was markedly different. In normal erythrocytes, pyrroline-5-carboxylate treatment increased pentose shunt activity 600%, PP-ribose-P formation 250%, and the incorporation of hypoxanthine into inosine monophosphate 260%. In contrast, pyrroline-5-carboxylate had no effect on glucose-6-phosphate dehydrogenase-deficient erythrocytes. These findings strongly support our proposed mechanism for the pyrroline-5-carboxylate effect on nucleotides. Furthermore, the markedly different capacities for nucleotide synthesis in the two populations with pyrroline-5-carboxylate treatment suggest a role for pyrroline-5-carboxylate-mediated modulation of nucleotide metabolism in normal cells.