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The model legume plant Medicago truncatula accumulates free proline in response to hyperosmotic stress as do many other organisms. In order to analyse the transcriptional regulation of proline biosynthesis in M. truncatula, three cDNAs encoding Δ¹‐pyrroline‐5‐carboxylate synthetase (P5CS1, P5CS2; EC not assigned) and ornithine δ‐aminotransferase (OAT; EC 2.6.1.13) were isolated. The cDNAs shared high homologies with the other plant sequences and genomic organization analysis indicated the presence of two P5CS and two putative OAT genes. The two P5CS genes showed differing transcript level regulation according to organs and in response to osmotic stress. MtP5CS1 steady‐state transcript levels in the different plant organs were correlated with proline levels but transcript abundance was unaffected by osmotic stresses. MtP5CS2 transcripts were poorly detected in all organs but were strongly …