Molecular investigations of a 37 yr old man and his family were undertaken to identify the molecular defect in a severe form of Osteogenesis Imperfecta(Type III), a hereditable connective tissue disorder caused by mutations in either of the genes coding for the two chains of type I collagen. SSCP analysis localized the mutation in the aa 579-679 subregion of α1(I). The sequence of the cDNA revealed a deletion of the last 3 nucleotides of exon 33/34 and the entire exons 35 and 36, with an insertion of 156 nt from 3′end of intron 36. The sequence of the genomic DNA contained the identical deletion. RT-PCR of the regions coding for aa 488-856 and 756-856 ofα1(I) chain identified two alternative splices of the mutant RNA in which the use of different donor sites results, respectively, in the lost of the last 5 nt of the deleted exon 33/34 (out-of-frame) and in the exon skipping of exon 33/34 (in-frame). The in-frame transcript is thus a deletion of the exons 33/34, 35 and 36. We used an RNA protection assay to quantify the proportion of mutant α1(I) transcripts in each of the three forms. We found that 2-7% of the transcript was in the retained intron form, 35-40% used the out-of-frame cryptic donor in exon 34 and 55% of the mRNA was in-frame with the exons 33/34, 35, 36 deleted. Biochemical analysis of the collagen synthesized from cultured fibroblasts of the patients were performed in an attempt to detect the mutant protein. We were unable to demonstrate its presence either in cells or in media. SDS-PAGE of type I procollagen produced by cultured osteoblasts of the probands showed normally migrating type I collagen chains and a higher than normal proportion of type III procollagen. The absence of detectable mutant protein and the increasedα1(III)/α1(I) ratio, which are charateristic of the mild form of OI, could moderate the lethal phenotype previously associated with the presence of large deletions in COL1A1 or COL1A2.