We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the α 1(1) and α2(l) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire proα1(l) and proα2(l) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for α1 (I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A + )-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes.We have used this strategy for the detection and sequencing of a point mutation in α2(1) mRNA associated with a case of lethal osteogenesis imperfecta. The G→A point mutation in the codon for α2(l) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.