<jats:p>The biosynthesis of the lipopeptide antibiotic surfactin was studied in whole cells of <jats:italic>Bacillus subtilis</jats:italic> ATCC 21332 which incorporate <jats:sup>14</jats:sup>C‐labeled precursor amino acids directly into the product. [<jats:sup>14</jats:sup>C]Acetate appeared in the fatty acid portion of surfactin and was also partially converted into leucine. An enzyme was isolated and partially purified from a cell‐free extract of the bacillus which catalyzes ATP‐P<jats:sub>i</jats:sub>‐exchange reactions which are mediated by the amino acid components of surfactin. This activation pattern is consistent with a peptide synthesizing multienzyme which activates its substrate amino acids simultaneously as reactive aminoacyl phosphates.