Strains of the sweet potato soil rot pathogen Streptomyces ipomoeae had previously been divided into three groups based on their ability to inhibit one another during pairwise cocultivation. While group I strains are not antagonistic to members of the other groups, group II and group III strains produce separate substances that are inhibitory to strains outside their respective cognate groups. Here, we purified the group III inhibitory substance from the culture supernatant of a representative strain and found that it consists of a single 10-kDa cationic protein which is bacteriolytic for S. ipomoeae group I and II strains but which showed no inhibitory function against other streptomycetes or other bacterial genera tested. The structural gene for the inhibitor was cloned from a chromosomal library of the producing strain, and while the gene sequence revealed that the inhibitor is initially made in a larger precursor form, the deduced mature protein showed no significant homology to other known proteins. Our results demonstrate that S. ipomoeae group III inhibitory activity is manifested in the form of a highly specific, potentially novel bacteriocin, which we have designated ipomicin.