We have isolated several cDNA clones encoding ^{1 1}-pyrroline-5-carboxylate reductase (P5CR, l-proline: NAD(P)⁺ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encoded a 28 586 M_r polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.