Transformants of S. lividans TK24 were obtained by transforming a recombinant plasmid p6BC12 harboring the thienamycin cyclase gene into it. An antibacterial substance could be detected by the conversion of fermentation broth of the Y3 block mutant and the purified Y3 mutant intermediate with a cell-free extract of S. lividans TK24 transformant. Paper chromatographic analysis showed that the conversion product of Y3 fermentation broth with cell-free extract of S. lividans TK24 was thienamycin and an unstable antibacterial substance was a product of Y3 intermediate with cell-free extract of the transformants. This result indicated that the thienamycin cyclase gene was expressed in S. lividans TK24 and complemented the deficiency in the Y3 block mutant. Restriction analysis of p6BC12 was carried out and the restriction map was constructed. The thienamycin cyclase gene was localized on a 0.9 kb PstI-HinCII fragment from a bioconversion result. The 1.0 kb IPNS homologous DNA fragment in plasmid p6BC12 was excluded from the cyclase activity.