The purpose of this study was to conduct a multidisciplinary analysis of a specific type of tooth enamel disturbance (amelogenesis imperfecta) affecting two Chilean families to obtain a precise diagnosis and to investigate possible underlying mutations. Two non-related families affected with amelogenesis imperfecta were evaluated with clinical, radiographic and histopathological methods. Furthermore, pedigrees of both families were constructed and the presence of eight mutations in the enamelin gene (ENAM) and three mutations in the enamelysin gene (MMP-20) were investigated by PCR and direct sequencing. In the two affected patients, the dental malformation presented as soft and easily disintegrated enamel and exposed dark dentin. Neither of the affected individuals presented with a dental and skeletal open bite. Histologically, a high level of an organic matrix with prismatic organization was found. Genetic analysis indicated that the condition is autosomal recessive in one family and either autosomal recessive or due to a new mutation in the other family. Molecular mutational analysis revealed that none of the eight mutations previously described in the ENAM gene or the three mutations in the MMP-20 gene were present in the probands. A multidisciplinary analysis allowed for a diagnosis of hypocalcified amelogenesis imperfecta, Witkop type III, which was unrelated to previously described mutations in the ENAM or MMP-20 genes.