A new member of the UDP-N-acetylglucosamine:␤-galactose ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) family having the ␤3Gn-T motifs was cloned from rat and human cDNA libraries and named ␤3Gn-T5 based on its position in a phylogenetic tree. We concluded that ␤3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc 3 Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto-or neolacto-series carbohydrate chains on glycolipids. ␤3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc 4 Cer; paragloboside), resulting in the synthesis of Lc 3 Cer and neolactopentaosylceramide (nLc 5 Cer), respectively. A marked decrease in LacCer and increase in nLc 4 Cer was detected in Namalwa cells stably expressing ␤3Gn-T5. This indicated that ␤3Gn-T5 exerted activity to synthesize Lc 3 Cer and decrease LacCer, followed by conversion to nLc 4 Cer via endogenous galactosylation. The following four findings further supported that ␤3Gn-T5 is Lc 3 Cer synthase. 1) The ␤3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc 3 Cer synthase in those cells. 2) The ␤3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The ␤3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in ␤3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc 3 Cer synthase activity reported previously.To date, three members of the human ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) 1 family (␤3Gn-T2, -T3, and -T4) (1, 2) and five members of the human ␤1,3-galactosyltransferase (␤3Gal-T) family (␤3Gal-T1, -T2, -T3, -T4, and -T5) have been identified (3-6). All of them share amino acid motifs (␤3Gn-T motifs or ␤3Gal-T motifs) in three regions of the catalytic domain. The first, ␤3Gn-T, was cloned by an expression cloning method using an anti-i antibody (7). However, this enzyme is unique in that it does not have the ␤3Gn-T motifs although it transfers GlcNAc to Gal with an ␤1,3-linkage, resulting in the synthesis of polylactosamine chains. It was named iGn-T (7). Thereafter, ␤3Gn-T1 was isolated based on structural similarity with the ␤3Gal-T family (2). We previously reported three additional ␤3Gn-Ts, ␤3Gn-T2, -T3, and -T4, which are also structurally related to the ␤3Gn-T family (1). However, the cDNA sequence of ␤3Gn-T1 was recently corrected by Zhou et al. (see Ref. 2). The corrected sequence of ␤3Gn-T1 was identical to that of ␤3Gn-T2 which was isolated * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with the accession num...