A cDNA, Atcys-3A, encoding O-acetylserine-(thiol)lyase has been isolated from Arabidopsis thaliana. The deduced peptide sequence showed a high level of similarity with the bacterial counterpart, and a remarkable percentage of identity with other higher plant O-acetylserine(thiol)lyase genes. Sequence comparison and Southern blot analysis suggested that Atcys-3A was a new and different to the previously reported member of the cytosolic gene family in Arabidopsis. The Atcys-3A expression was activated by sulfur limitation, requiring a carbon and nitrogen source for maximal expression. A similar pattern of regulation was observed at the O-acetylserine(thiol)lyase activity level. Northern blot analysis also showed an organ-specific expression of Atcys-3A.