The purpose of this report was to investigate by two independent means the status of the tetrafunctional cross-link dehydrohistidinohydroxymerodesmosine in collagen fibrils and to locate in the protein molecule the contributing histidine residue. These aims are consistent with obtaining insights into the three-dimensional packing of collagen molecules in fibrils. Insoluble collagen fibrils treated at pH 4.3 to break the crosslink into its precursors were re-equilibrated at pH 7.4, 37” C, to allow re-formation of dehydrohistidinohydroxymerodesmosine. It was demonstrated that there was a time-dependbnt increase in its re-formation which reached control values at 24 h. The data were consistent with the Michael addition of the N of histidine across the ethylene double bond of the a,/3 unsaturated aldehyde, aldol condensation product, the interchain intramolecular cross-link of collagen. Iodination of purified reconstituted soluble guinea pig skin collagen fibrils at pH 7.4 destroyed all but 1 to 2 histidine residues/mol of collagen with full retention of dehydrohistidinohydroxymerodesmosine. This indicated that the N of the histidine residue participating in the cross-link was protected from modification by alkylation as the Michael adduct. Pepsin solubilization of the iodinated fibrils, denaturation, gel filtration, and 0-(carboxymethy1) cellulose chromatography allowed isolation of two a1 chains. Chromatographic differences were probably due to fractional site participation of imidazole with iodine. Preparative polyacrylamide gel electrophoresis of CNBr digests of the two a1 chains isolated from the modified fibrils allowed preparation of alCB6-8 and alCB7, G from each. These CNBr peptides contain the histidines present in al. Amino acid analyses indicated 1 residue of histidine in each of alCB6-8 peptides and a fractional residue in each preparation of alCB7, 6. It was surmised, therefore, that histidine 89 of the a1 chain participates in the formation of histidinohydroxymerodesmosine obtained from NaBI&-reduced collagen fibrils. It was concluded from the reformation study and the iodine modification work that dehydrohistidinohydroxymerodesmosine is a real and natural crosslink in collagen fibrils and not an artifactual compound catalyzed by the borohydride or cyanoborohydride anions in the reduction procedure as claimed by Robins