Attempts were made to optimise protoplast formation and regeneration methods to improve the protoplast fusion frequencies of Streptomyces coelicolor. The yields of protoplast formation and regeneration were varied with different growth phase of the culture. Maximum yields were obtained when cells were taken from the late logarithmic phase. Protoplast formation reached almost its maximum with lysozyme treatment at a concentration of 2mg/ml without any other lytic enzyme. A high frequency of protoplast regeneration was accomplished by overlay method: the method gave 14% recovery of regenerated protoplast versus 1.8% recovery for monolay method. A recombinant frequency of 1.8X10^-2 was obtained by protoplast fusion using PEG 1000(50% w/v).