Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.