Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers 3H from [1-3H]glucose to NADP+ to delta 1-pyrroline-5-carboxylate and yields [3H]proline. The formation of [3H]proline depends on the presence of NADP, delta 1-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and delta 1-pyrroline-5-carboxylate reductase. The production of [3H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to delta 1-pyrroline-5-carboxylate by mitochondrial proline oxidase. delta 1-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by delta 1-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of delta 1-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.