When cells of Escherichia coli are grown on lactate (or other carbon sources), an addition of serine to the medium causes growth inhibition. This growth inhibition is caused by inhibition by serine of homoserine dehydrogenase I, which is involved in threonineisoleucine biosynthesis [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. We have cloned and sequenced genes which enhance the serine-sensitivity. Two open reading frames were found and designated as sseA and sseB. Introduction of either sseA or sseB gene, or both, into E. coli cells enhanced the serine-sensitivity. The sseA gene elicited stronger enhancement than sseB. The deduced amino acid sequence of SseA showed considerable similarity with that of bovine liver rhodanese, which catalyzes sulfur transfer from thiosulfate. We observed a twofold increase in rhodanese activity in E. coli cells harboring a plasmid carrying the sseA gene. The position of sseA in the genetic map is around 52'. However, sseA is different from cysM, which codes for O-acetylserine sulfhydrylase-B, an enzyme catalyzing sulfur transfer from thiosulfate to O-acetylserine, the map position of which is also around 52'.