delta 1-Pyrroline-5-carboxylic acid, an intermediate in the interconversions of proline, ornithine, and glutamate, is a potent stimulator of glucose oxidation through the hexosemonophosphate-pentose pathway. The effect is observed in cultured human fibroblasts, Chinese hamster ovary cells (CHO-K1), and rabbit kidney cells (LLC-RK1). In human fibroblasts, the magnitude of the stimulation of the hexosemonophosphate-pentose pathway is dependent on the concentration of added pyrroline-5-carboxylate and the effect is observed over a wide range of glucose concentrations. The mechanism of the effect is related to the generation of oxidizing potential in the form of NADP+ by pyrroline-5-carboxylate reductase concomitant with the conversion of pyrroline-5-carboxylate to proline. In LLC-RK1 cells, a cell line unique in having proline oxidase activity, proline also stimulated hexosemonophosphate-pentose pathway activity. Although pyrroline-5-carboxylate markedly stimulated the hexosemonophosphate-pentose pathway, it has no effect on glucose metabolism in the Embden-Meyerhof pathway or the tricarboxylic acid cycle. Since the hexosemonophosphate-pentose pathway is a source of ribose-5-phosphate, the precursor of phosphoribosyl pyrophosphate, the effect of pyrroline-5-carboxylate on the hexosemonophosphate-pentose pathway may link amino acid and nucleic acid metabolism.