gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.