For improvement of the regeneration frequency of Streptomyces griseus 2247 protoplasts, the effects of growth phase and components of the regeneration medium on the frequency were investigated. Protoplasts prepared from transition-phase mycelia of S. griseus 2247 exhibited the highest regeneration frequency. An improved regeneration medium designated R1M was established by supplementing Polypepton and yeast extract which were shown to increase the S. griseus 2247 growth rate to R1 previously developed for S. griseus. The regeneration frequencies of S. griseus 2247 and its pleiotropic mutant HT-3 were greatly increased to 87.5% and 92.5%, respectively, on R1M medium. Using this protoplast-regeneration system, the transformation efficiency for the plasmid pRES18 was increased to 10(6) transformants per mu g DNA. R1M medium was also effective for the protoplast regeneration of some other antibiotic-producing streptomycete species.