BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:β-N-acetyl-glucosamine β-1,3- galactosyltransferase, designated β3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to β3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP- galactose:β-N-acetyl-glucosemine β-1,3-galactosyltransferase (β3Gal-T2) with similar kinetic properties as β3Gal-T1. Another gene represented a UDP- galactose:β-N-acetyl-galactosamine β-1,3-galactosyltransferase (β3Gal-T4) involved in G(M1)/G(D1) ganglioside synthesis, and this gene was highly similar to a recently reported rat G(D1) synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. β3Gal-T1 mRNA was expressed in brain, β3Gal-T2 was expressed in brain and heart, and β3Gal- T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. β3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous β3- galactosyltransferase genes.