The L-proline-dependent reduction of NAD+ has been obtained with a soluble enzyme extracted from acetone powders of the cotyledons of 3- to 5-day-old germinating peanut seedlings. The enzyme has been purified approximately 20-fold. NAD+ is much more effective as an electron acceptor than NADP+, the reaction rate with the latter being only 15 per cent that with the former. The Km for L-proline at pH 10.3, with NAD+ saturating, is 0.30 mM, and that for NAD+, with L-proline saturating, is 0.25 mM. NADP+ is an excellent competitive inhibitor for NAD+ with a K1 of 6.2 μM. L-proline, L-proline methyl ester, and 3,4-dehydro-DL-proline are equally effective as substrates. Thiazolidine-4-carboxylate catalyses the reduction of NAD+ at 63 per cent the rate with L-proline. D-proline is not a substrate nor an inhibitor. L-proline amide has 11 per cent the activity of L-proline and N-methyl-L-proline has a very slight activity. Other proline derivatives or the lower and higher homologues are completely inactive. Incubation with L-proline-14C in the presence of NAD+ yields one product which has a higher Rf than proline using butanol-acetic acid-water as the solvent in paper chromatography. Elution of this product and treatment with hydrogen peroxide gives several products of high Rf with the same solvent mixture. None of the products is γ-aminobutyrate or glutamic acid. This eliminates either P2C or P5C as the reaction product.