Skin fibroblasts from two affected members of a family with an autosomal dominant form of mild-moderate osteogenesis imperfecta produced two populations of type I collagen molecules. One population was normal and the other population contained alpha 2(I) chains which had a basic charge shift localized to a peptide from the carboxyl-terminal end of the triple-helical domain. The alpha chains in the abnormal molecules had increased post-translational modification along the entire triple-helical domain but the thermal stability was normal. We isolated a 28-kb BamHI fragment from the normal and mutant COL1A2 alleles from an affected family member. DNA sequence determination demonstrated that a single nucleotide change resulted in an arginine for glycine substitution at triple-helical position 1012, the last triple-helical glycine. These data demonstrate the stringent requirement for maintenance of the Gly-X-Y triplet sequence in type I collagen and suggest that point mutations which disrupt Gly-X-Y in alpha 2(I) produce milder clinical effects than similar mutations in alpha 1(I).