Skin fibroblasts from a proband with lethal osteogenesis imperfecta synthesized a type I procollagen containing a cysteine residue in the alpha 1(I) helical domain. Assay of thermal stability of the triple helix by proteinase digestion demonstrated a decreased temperature for thermal unfolding of the protein. Of special importance was the observation that assays of thermal stability by proteinase digestion revealed two bands present in a 2:1 ratio of about 140 and 70 kDa; the 140 kDa band was reducible to a 70 kDa band. Further analysis of the fragments demonstrated that the cysteine mutation produced a local unfolding of the triple helix around residue 700 and apparently exposed the arginine residue at position 704 in both the alpha 1(I) and alpha 2(I) chains. Analysis of cDNAs and genomic DNAs demonstrated a single-base mutation that changed the GGT codon for glycine-691 of the alpha 1(I) chain to a TGT codon for cysteine. The mutation was not found in DNA from either of the proband's parents. Since the proteinase assay of helical stability generated a fragment of 700 residues that retained disulphide-bonded cysteine residues at alpha 1-691, the results provide one of the first indications that glycine substitutions in type I procollagen can alter the conformation of the triple helix at a site that is C-terminal to the site of the substitution.