The thymidylate synthase (TS) gene from Lactobacillus casei has been isolated, cloned, and sequenced. The coding sequence is 948 bp and predicts a primary structure, identical to that reported by protein sequencing methods (Maley et al., 1979b). The gene has been placed in several expression systems which complement TS-deficient Escherichia coli, and express the catalytically active enzyme at levels of 10-20% of the soluble protein of E. coli. The expressed TS has kinetic and structural properties consistent with its being identical to the authentic enzyme from L. casei.