Aims: To devise and evaluate a method for isolating che rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter. Methods and Results: The newly developed method consists of tno enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates. Conclusions: A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa. Significance and Impact of the Study: The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy; and bioactivity.