Osteogenesis imperfecta (OI) type I is characterized by bone fragility without significant deformity, osteopenia, normal stature, blue sclerae, and autosomal dominant inheritance. Dermal fibroblasts from most affected individuals produce about half the expected amount of type I collagen, suggesting that the OI type I phenotype results from a variety of mutations which alter the apparent expression of either COL1A1 or COL1A2, the genes encoding the chains of type I collagen. Short-pulse labeling of dermal fibroblasts with [3H]proline from affected individuals in 19 families indicates that most have alterations in the expected 2:1 synthetic ratio of pro alpha 1(I): pro alpha 2(I), with most having decreased production of pro alpha 1(I). Ratios of COL1A1:COL1A2 mRNA from these individuals, using slot-blot hybridization, indicate that they fall into different groups, but that most have decreased COL1A1 mRNA levels, compared with controls. These data suggest that most of our OI I families have COL1A1 mutations. Copy number and size of the COL1A1 gene by restriction endonuclease analysis of genomic DNA from affected individuals are normal in the families examined. We have identified one 3 generation family in which all affected members have one normal COL1A1 allele and another with a 5 base-pair deletion near the 3' end of the gene. The deletion creates a shift in the translational reading-frame and predicts the synthesis of an elongated pro alpha 1(I) chain. In a second family, a father and a son have a single exon deletion that results from a splicing mutation. Chemical cleavage analysis of amplified cDNA from affected individuals in different regions of the COL1A1 gene, including the promoter, suggests that several individuals have point mutations within the coding region of the gene, while one individual may have a small deletion within the alpha 1(I) carboxyl-terminal propeptide region. Our data provide evidence for significant molecular heterogeneity within the OI type I phenotype and indicate that a variety of mutations can result in decreased synthesis of type I collagen.