The accumulation of free L-proline in leaves as a response to water deficit is well documented. Accumulation is caused by stress enhanced-de novo proline synthesis from glutamic acid. Three enzymes, gamma-glutamyl kinase (GK, EC 2.7.2.11), gamma-glutamyl phosphate reductase (GPR, EC 1.2.1.41), and pyrroline-5-carboxylate reductase (PCR, EC 1.5.1.2) catalyze the pathway in other organisms;A continuous assay for GK was developed. A crude preparation from etiolated shoots of Vigna radiata L. was capable of synthesizing pyrroline-5-carboxylate (P5C) from glutamic acid. Attempts to purify GPR activity yielded P5C dehydrogenase activity that was not phosphate-dependent. PCR was the only enzyme whose activity could be measured;Protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated on a Percoll gradient. PCR activity was found in chloroplasts. PCR activity from pea chloroplasts and etiolated pea shoots was compared. PCR activity from both extracts is stimulated at least twofold by 100 mM KCl and 10 mM MgCl[subscript]2. The pH profiles of PCR activity from both extracts reveal two separate pH optima at 6.5 and 7.5. PCR activity from etiolated pea shoots was 100-fold greater than that from pea chloroplasts. Three nonionic detergents (NP-40, CHAPS, and CHAPSO) doubled the yield of PCR activity from pea chloroplasts;PCR was partially purified from pea leaf chloroplasts and from etiolated pea shoots. PCR activity from chloroplasts exhibited a preference for NADPH. PCR activity from etiolated shoots had a higher K[subscript] m for NADH than for NADPH. The V[subscript] max was also greater with NADH than with NADPH. PCR activity from etiolated shoots was inhibited by 50 mM proline, 2 mM azetidine-2-carboxylate, and 2 mM thiazolidine-4-carboxylate. The molecular mass of native PCR was greater than 100 kd. The pI of native PCR was 7.8. Participation of PCR in the regulation of metabolism during stress is indicated.