The most common form of beta-thalassemia among Mediterraneans results from a single nucleotide substitution within the first intervening sequence (IVS-1) of the beta-globin gene. This particular mutation is not detectable in uncloned DNA by restriction enzyme analysis. Using synthetic DNA of 19-nucleotides in length corresponding to the normal and mutant IVS-1 sequences as probes, we have developed a direct assay for this gene defect. Under carefully controlled experimental conditions these synthetic probes detect only their homologous sequences in restriction digests of both cloned and uncloned DNA samples. The method is sufficiently sensitive to establish the genotype of individuals with respect to this defect using approximately 20 micrograms total DNA. This assay provides an alternative to fetal blood and DNA linkage analysis for the prenatal diagnosis of this variety of beta-thalassemia, particularly among Greek families where it is especially common.