A quantitative real-time PCR method was developed and used for determination of streptomycetes in indoor dust samples of five homes collected during three years. The specificity of the method was tested with 14 Streptomyces and ten non-streptomycetous species, revealing a high specificity for mesophilic streptomycetes. Thermophilic species and S. albus were not efficiently detected. The method gave reproducible results in replicate analyses of the same dust DNA as well as of duplicate DNA isolations. The amount of streptomycetes in house dust was lowest in winter, followed by summer, and highest in spring and fall. The greatest variation in Streptomyces-concentrations was observed in the spring and fall samples.