Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.