Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of ¹H homonuclear and ¹H-¹³C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be → 3)-β-d-Glcp-(1 → 3)-α-d-Galp-(1 → 3)-β-d-Galp-(1 → as opposed to → 3)[α-d-Galp-(1 → 6)]-β-d-Glcp-(1 → 3)-α-d-GalpNAc-(1 → 3)-β-d-Galp-(1 → for the wild-type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate.