Fibrous collagen from normal (line 412) and dystrophic (line 413) chicken pectoral muscles was examined for susceptibility to bacterial collagenase by a pH-stat titration method to assess the kinetics of peptide bond hydrolysis. These experiments showed that there was a profound difference in the kinetics of hydrolysis using fibrous collagen purified from normal and dystrophic muscle. Although normal collagen was readily hydrolyzed by the collagenase, dystrophic collagen was not hydrolyzed in the presence of 200 FM Ca and was hydrolyzed slowly in the presence of 5 mM Ca. At this high Ca concentration, the K, for collagenase was six times higher in dystrophic collagen than in normal, whereas the maximum reaction rate (VmaX) remained the same. After CNBr digestion, peptides from both sources were readily hydrolyzed by collagenase in the presence of 200 FM Ca, and there was no noticeble difference in the reaction kinetics. The above results were not modified whether the chicken was sacrificed on day 4 or day 98 posthatching, and the insusceptibility to collagenase preceded the clinical manifestations of dystrophy. The structure of both fibrous and CNBr-treated collagen from normal and dystrophic muscle was compared. One and twodimensional gel electrophoreses showed identical peptide maps. Gel filtration of CNBr peptides showed in the excluded volume the presence of twice as much high-molecular-weight (presumably crosslinked) peptides from dystrophic collagen compared with normal. We conclude that the intermolecular crosslinks are more extensively formed in dystrophic collagen and that this increased crosslinking results in the increased resistance of the dystrphic collagen to collagenase.