Mutations in COL1A1 and COL1A2 genes, encoding the α1 and α2 chain of type I collagen, respectively, are responsible for the vast majority of cases of osteogenesis imperfecta (OI) (95% of patients with a definite clinical diagnosis). We have investigated 22 OI patients, representing a heterogeneous phenotypic range, at the biochemical and molecular level. A causal mutation in either type I collagen gene was identified in 20 of them: no recurrent mutation was found in unrelated subjects; 15 out of 20 mutations had not been reported previously. In two patients, we could not find any causative mutation in either type I collagen gene, after extensive genomic DNA sequencing. Failure of COL1A1/COL1A2 mutation screening may be due, in a few cases, to further clinical heterogeneity, i.e. additional non-collagenous disease loci are presumably involved in OI types beyond the traditional Sillence's classification.