The O-acetylhomoserine sulthydrylase (OAHS) gene was cloned from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library by degenerative probe hybridization and complementation. Sequence analysis revealed an 869-bp ORF with a G + C content of 53%. The ORF had significant homology with enzymes involved in homocysteine biosynthesis. A CuraBLASTN homology search showed that the ORF has 63% nucleotide identity with the OAHS of Bacillus stearothermophilus, Corynebacterium glutamicum, and Acremonium chrysogenum, and has 58% identity with met25 of Saccharomyces cerevisiae and metZ of Pseudomonas aeruginosa. The deduced amino acid sequence exhibited 45% similarity with Met25 and MetZ. Further analysis predicted that the gene product was a member of the pyridoxal phosphate enzyme family. Complementation experiments with Escherichia coli metA, metB, and metC mutant strains showed that the S. ruminantium OAHS gene can complement the metC mutation and allow for growth on minimal media that contained sodium thiosulfate as the sole source of sulfur. When the OAHS was disturbed by inserting a EZ::TN pMOD-2 transposon, the complementation was lost. Therefore, these results suggest that the gene functions as OAHS in S. ruminantium HD4.