Spiramycin-producing Streptomyces ambofaciens KA-1028 harboring the pSA1 plasmid gave rise to spiramycin non-producing variants at high frequencies by various curing treatments. However, a number of the spiramycin non-producing progeny obtained by treatment with acridine dyes, still harbored plasmid DNAs which could not be differentiated from plasmid pSA1 by contour length, cleavage patterns and heteroduplex analysis. By treatment with mitomycin C, plasmid pSA1 was cured at high efficiency and spiramycin non-producing strains were obtained. Strain U-1717R obtained by regeneration of protoplasts of plasmid-cured strain U-1717 regained spiramycin production on growth on solid medium only. Furthermore, transconjugants obtained by mating between strain KA-1028 and U-1717R-24 (streptomycin-resistant) regained spiramycin production in both liquid and solid media. We conclude that the genes for the biosynthesis of spiramycin are encoded in a replicon other than plasmid pSA1 but that this plasmid plays a role in the regulation of spiramycin production.