Herbicolin A, a novel acyl peptide antibiotic, inhibits the growth of the sterol-requiring Mycoplasma, Ureaplasma, and Spiroplasma species, with MICs varying from 1.5 to 100 micrograms/ml. With the exception of Acholeplasma modicum, all of the non-sterol-requiring species of the family Acholeplasmataceae were totally resistant to herbicolin A when tested on serum-containing medium but were inhibited to some extent on medium devoid of serum and any other source of sterol or fatty acids.