A full‐length cDNA and the corresponding At‐P5S gene encoding the first enzyme of the proline biosynthetic pathway, the Δ ¹‐pyrroline‐5‐carboxylate (P5C) synthetase, were isolated in Arabidopsis thaliana. The At‐P5S cDNA encodes a protein of 717 amino acids showing high identity with the P5C synthetase of Vigna aconitifolia. Strong homology is also found at the N‐terminus to bacterial and yeast γ‐glutamyl kinase and at the C‐terminus to bacterial γ‐glutamyl phosphate reductase. Putative ATP‐ and NAD(P)H‐binding sites are suggested in the At‐P5S protein. The transcribed region of the At‐P5S gene is 4.8 kb long and contains 20 exons. Southern analysis suggests the presence of only one At‐P5S gene in the A. thaliana genome mapped at the bottom of the chromosome two. Expression analysis of At‐P5S in different organs reveals abundant At‐P5S transcripts in mature flowering plant. Rapid induction of the At‐P5S gene followed by accumulation of proline was observed in NaCl‐treated seedlings suggesting that At‐P5S is osmoregulated.