Type I collagen mutations in a group of patients with lethal perinatal osteogenesis imperfecta were identified in fibroblast RNA by a new method which can detect, by chemical modification and cleavage, single mismatched bases in heteroduplexes formed between mRNA and normal cDNA probes. Control cDNA probes spanning the area of the pro-alpha 1(I) and pro-alpha 2(I) chains likely to contain the mutations were radioactively labeled and used to form heteroduplexes with total patient RNA. Treatment of these heteroduplexes with hydroxylamine followed by cleavage of the cDNA strand at reactive bases by piperidine identified mismatches in the pro-alpha 1(I) cDNA in four patients. In the fifth patient a mismatch was detected in the pro-alpha 2(I) cDNA. To characterize these mutations the regions containing the mismatches were amplified by the polymerase chain reaction, cloned, and sequenced. All were heterozygous single base mutations which led to the substitution of glycine residues in the helical region of the pro-alpha-chains. The substitutions were pro-alpha 1(I) Gly973 and Gly1006 to Val, Gly928 to Ala, Gly976 to Arg, and pro-alpha 2(I) Gly865 to Ser. These mutations emphasize the importance of the Gly-X-Y repeating amino acid sequence for normal collagen helix formation and function in the extracellular matrix.