From a systematic search of the UniGene and dbEST databanks, using human β4-galactosyltransferase (β4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as β4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to β4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (β4GalT-VI) to 55% (β4GalT-II). The highest sequence identity between any of the homologs is between β4GalT-V and β4GalT-VI (68%). β4GalT-II is the ortholog of the chicken β4GalT-II gene, which has been demonstrated to encode an α-lactalbumin responsive β4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, β4GalT-II and -VI show the most restricted pattern of tissue expression. High steady state levels of β4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of β4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the β4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the β4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known β4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the β4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.