Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.