Antibiotic production, formation of aerial mycelia (Amy), and nutrient auxotrophy of some streptomycetes have been reported to be related to the loss of plasmids, the movement of a transposable element, or the appearance of reiterated DNA sequences in the bacterial chromosome. We have been engaged in an investigation to analyze in detail these relationships in streptomycetes which produce polyether antibiotics. During the course of these studies, we found that when Streptomyces hygroscopicus 358AV2, a carriomycin producing strain, was subjected to protoplast regeneration, the regeneration rate was as high as 90%, and it lost the ability to produce the antibiotic at a high frequency (about 90%) with the formation of aerial mycelium being unaffected. Protoplast regeneration has been reported to result in the loss of plasmids of streptomycetes and alteration of several phenotypes; for example, the pSV1 plasmid, which codes for synthesis of and resistance to methylenomycin A, was eliminated by protoplast regeneration, suggesting that carriomycin non-producing mutants are formed by the loss of plasmid(s) or the deletion of DNA during protoplast regeneration. Treatment of the parent strain with ethidium bromide gave Amy- mutant strains retaining the productivity of carriomycin at a high frequency (about 70%), but no carriomycin non-producing mutants. The productivity of carriomycin in these mutant strains was inferior to that of the parent strain. Except for the formation of aerial mycelium, most of these mutants had the same phenotypic properties as the parent strain. Surprisingly, however, half of these mutants produced an unknown antibacterial substance in addition to carriomycin. In this paper we wish to report the isolation and structural elucidation of this antibiotic, which we have named curromycin A.