To understand the role of type V collagen and its spatial interrelationship with type I collagen in bone matrix, the molecule's covalent intermolecular cross-links were structurally characterized. Type V collagen containing α1(V), α2(V) and α1(XI) chains was isolated from bovine bone and reacted with NaB³H₄ to label the cross-linking residues. Radiolabeled native molecules and isolated a chains were treated with sodium metaperiodate to cleave the divalent cross-linking bonds. Sequence analysis of the periodate-released peptides matched two of them to α1(V) and α1(XI) aminopropeptide domains. A third peptide was derived from the α1(I) carboxytelopeptide domain of type I collagen. This latter peptide, therefore, came from a site of heterotypic cross-linking between types I and V collagens and accounted for about 15% of the total cross-linked peptides. Sequence analysis of isolated cross-linked tryptic peptides defined the helical sites of attachment of the periodate-released telopeptides and revealed that the putative aminoproteinase-cleavage sites in the α1(V) and α1(XI) chains are located in the molecule interior to the cross-linking residue. These data imply that type V collagen molecules in the extracellular matrix are primarily cross-linked to each other in a head-to-tail linear polymer that is linked laterally to type I collagen molecules in copolymeric fibrils.