-Glutamylcysteine synthetase (-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report we show that crude extracts of Streptococcus agalactiae catalyze the -GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae -GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L--glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L--glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as -GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine and transition-state analog sulfoximines. The N-terminal 518 amino acids of -GCS-GS (total Mr 85 kDa) show 32 % identity and 43 % similarity with E. coli -GCS (Mr 56 kDa), but the C-terminal putative GS domain (remaining 202 amino acids) of -GCS-GS shows no significant homology with known GS sequences. The C-terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24 % identity; 38 % similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.