Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)_n triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host–guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)₃-Zaa-Pro-Hyp-(Gly-Pro-Hyp)₄-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T_m = 45°C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T_m decreases to ≈10°C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T_m < 0°C. A Gly → Cys replacement results in T_m < 0°C under reducing conditions but shows a broad transition (T_m ≈ 19°C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T_m by ≈5°C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala ≤ Ser < CPO_red < Arg < Val < Glu ≤ Asp. The rank of destabilization of substitutions for Gly in these Gly-Pro-Hyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the α1 chain of type I collagen.