Three macrolide antibiotic-producing strains, the spiramycin producer Streptomyces ambofaciens, the tylosin producer Streptomyces fradiae, and the cirramycin producer Streptomyces cirratus easily formed protoplasts when treated with lysozyme in hypertonic medium. Each type of protoplast was regenerated to a mycelial form at a frequency of 90 to 100% in regeneration medium supplemented with a plasma expander. In the spiramycin producer S. ambofaciens and the tylosin producer S. fradiae, antibiotic productivities of the regenerated progeny were drastically changed. Some regenerated progeny from the three strains gave higher production. One from S. ambofaciens showed about twice the productivity of the original strain and one obtained after two rounds of protoplast regeneration from S. fradiae produced about three times as much as the original strain.