We used the polymerase chain reaction (PCR) and allele-specific-oligonucleotide hybridization (ASO) or restriction enzyme analysis (RE) to investigate the molecular defect in 100 Italian subjects heterozygous for beta-thalassemia, members of 50 couples at risk for the disease: 93 out of the 100 alleles studied were identified after investigating 9 known mutations. The mutation was identified in both members of the couple in 43/50 cases; in the remaining 7 couples the defect was identified only in one subject. The PCR-ASO or-RE method is a suitable, though still complex, approach to prenatal diagnosis of beta-thalassemia in a population with heterogeneous molecular defects.