Procollagen synthesized by skin fibroblasts from a patient with a lethal variant of osteogenesis imperfecta has been characterized. After pepsin digestion of the type I procollagen, a portion of the alpha 1(I) chains was recovered as a disulfide-bonded dimer. Cyanogen bromide peptide mapping suggested that a new cysteine residue was present in the alpha 1(I)CB6 fragment. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 946 of the alpha 1(I) chain to a cysteine codon. The thermal stability of the molecules was markedly lower than that in the case of the normal control.