The streptomycin (SM) gene cluster was investigated for its distribution in streptomycetes by Southern hybridization using nick-translated DNA probes, which were isolated from the SM-6-phosphotransferase (SPH) and amidinotransferase (ADT) regions of the SM gene cluster of Streptomyces griseus SS-1198. Bgl II-digested genomic DNAs from SM-producing strains of S. griseus yielded the same size fragment (7.0 kb) which hybridized to both the SPH and ADT probes as expected from the restriction endonuclease cleavage map of the SM gene cluster. By contrast, no genomic DNA fragments from heterologous Streptomyces strains hybridized to the probes. Thus, only SM-producing strains of S. griseus possess the highly homologous SM gene cluster. Similarly, distribution of DNA sequences homologous to the kanamycin (KM)-resistance determinant (kan) from a KM-resistant regenerant of S. griseus SS-1198 protoplasts was also examined. Using the kan gene fragment as the probe it was revealed that the kan-related sequences are present in all the strains of S. griseus tested, irrespective of the type of antibiotics they produce. However, no hybridization to the kan gene probe (KAN) was observed with DNA digests derived from other Streptomyces species.