Native collagen polypeptides exist in a unique triple helical conformation resistant to most proteinases. In this study, the stability of type I collagen triple helix, employing a mixture of trypsin and α-chymotrypsin as a proteolytic probe, was examined. The degradation of type I [ 3H]collagen was monitored as 3H-labeled peptides soluble in trichloroacetic acid (TCA) or by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis. In one set of experiments, collagen substrates were preincubated at various temperatures for up to 8 h, followed by a 15-min proteolytic treatment at the same temperature. At 43 °C, most of the collagen was degraded, while the fraction of the substrate degraded at 40, 38, and 35 °C was 53, 41 and 19%, respectively. This fraction was independent of the preincubation time which varied from 10 to 480 min. Thus, at any given temperature, a constant fraction of the collagen substrate was susceptible to proteolysis. Measurement of the midpoint temperature ( T m) of the helix to coil transformation for type I collagen, at neutral pH employing an increasing temperature gradient and brief proteolysis at the individual temperatures, indicated a value of 38.8 °C. However, determination of the T m by employing proteolytic digestions at a constant temperature (30 °C) using conditions under which the nonhelical peptides are readily digested to TCA-soluble peptides while native collagen resists such proteolysis, indicated a value of 42.7 °C. In further studies, collagen was subjected to continuous proteolysis for up to 24 h. A large fraction of collagen was digested at 30 or 34 °C, temperatures well below the T m of the helix to coil transformation. SDS-polyacrylamide gel electrophoresis of the degradation products obtained at these temperatures revealed multiple cleavage fragments. Finally, temperature double-jump experiments indicated that the destabilization of the triple helix is reversible provided that the T m of the substrate is not exceeded. The results provide evidence for reversible and local relaxation of the collagen triple helix.