The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.