Lysyl hydroxylase (LH), with three isoenzymes in vertebrates, catalyzes the formation of hydroxylysine by acting on -X-Lys-Gly- triplets in the collagenous domains of proteins of the collagen superfamily and also in -X-Lys-Ala- or -X-Lys-Ser- sequences in the telopeptides located at the ends of the polypeptide chains in some fibril-forming collagens. The hydroxylysine residues are essential for the stability of collagen crosslinks and act as carbohydrate attachment sites. The extent of lysine hydroxylation varies between collagen types, between tissues in the same collagen type and in certain diseases, suggesting that the LH isoenzymes may have different substrate specificities. We studied here the hydroxylation of synthetic peptides representing various hydroxylation sites in type I and IV collagens by purified recombinant LHs in vitro and of a recombinant full-length type I procollagen chain coexpressed with each LH in insect cells. All three LHs hydroxylated peptides representing collagenous sequences of type I and IV collagens, although with different K(m) and V(max) values. Furthermore, all three hydroxylated the collagenous domain of the coexpressed type I procollagen chain to a similar extent. None of the isoenzymes hydroxylated peptides representing the N and C telopeptides of type I collagen, but LH2, unlike the other two isoenzymes, hydroxylated the N telopeptide in the coexpressed procollagen chain. Hydroxylation of the telopeptide lysines by LH2 thus occurs only in the context of a long peptide. These data provide the first direct evidence that LH2 is a specific telopeptide hydroxylase, while all three LHs act on collagenous sequences.