From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-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 beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.