In the present study, a brain abundant member of beta 4-galactosyltransferase gene family with an open reading frame encoding 343 amino acids was cloned and identified from a human leukemia cell cDNA library. The putative protein sequence is about 94.8 and 94.2% identical to the 382-amino-acid mouse and rat beta 4-galactosyltransferase respectively and also contains cysteine residues previously shown to be important for the function of the gene family members. This cDNA (tentatively termed beta 4GalT-VIb) is identical to a recently reported cDNA (tentatively termed beta 4GalT-VIa) of human beta 4-galactosyltransferase except lacking one exon, suggesting that these two cDNAs are two different alternative transcripts of the same gene. Northern hybridization showed that the new alternative transcript, beta 4GalT-VIb, is expressed in all 16 human tissues tested with highest level in brain and rich level in testis, thymus and pancreas, whereas weak expression was detected in lung. The beta 4GalT-VIb gene was located to human chromosome 18q12.1 between markers WI-9180 and SGC35630 by radiation hybrid mapping. The genomic organization and adjacent gene content of beta 4GalT-VIb were identified by comparing its cDNA sequence with three genomic sequences AC017100, AP002474 and AP001336, which showed that beta 4GalT-VIb spans an approximately 58 kb region and is composed of 8 exons. In addition, the most conserved motif composed of 41 residues, LXYX3FGGVSXL(T/S)X2 QFX2INGFPNX(Y/F)WGWGGEDDDX2NR, was defined according to 17 sequences of beta 4GalTs from seven different organisms for the first time.