Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two alpha 1(I) chains and one alpha 2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the alpha 1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with alpha 2(I) homotrimers cleaved efficiently at a rate similar to that seen for alpha 1(II) and alpha 1(III) homotrimers, while alpha 1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the alpha 2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in alpha 2(I) homotrimers compared with alpha 1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that alpha 2(I) homotrimers are susceptible to MMP-1, proves that the presence of an alpha 1(I) chain is not a requirement for alpha 2(l) cleavage, and supports the importance of local unfolding of alpha 2(I) in collagenase cleavage.