We isolated a rice T-DNA tagging line, in which T-DNA was inserted into the sixth intron of OsP5CS2. This gene encodes for a protein that is highly homologous to 1-pyrroline-5-carboxylate synthetase (P5CS), a proline biosynthesis enzyme. The T-DNA contained the promoterless gus gene, allowing generation of a gene fusion between OsP5CS2 and gus. Therefore, the expression pattern of OsP5CS2 could be easily monitored by in situ GUS assay. At the seedling stage, the transcript level was low. However, gene expression was preferentially induced in the dividing zone of the roots by salt, cold, or ABA treatments. In mature spikelets, the gene was expressed mainly in stamens. RT-PCR analyses confirmed the results from the GUS assay. OsP5CS2 transcript was present in reproductive organs, especially the stamens. In seedling roots, transcript levels were increased by treatment with 250 mM NaCl, 4 ◦C cold stress, or 0.5 M ABA. Our OsP5CS2 knockout plants were more sensitive to salt and cold stresses than were the wild-type controls. Root and shoot growth in the knockout seedlings were severely retarded when plants were exposed to 250 mM NaCl. Cold treatment for more than 12 h also caused growth retardation in those seedlings. Therefore, our results indicate that the OsP5CS2 gene is necessary for plant tolerance to salt and cold stresses. © 2004 Elsevier Ireland Ltd. All rights reserved.