Acaterin (1), produced by Pseudomonas sp. A 92, is a secondary metabolite having a 2-penten-4-olide structure. Feeding experiments with (2)H- and (13)C-labeled decanoic acid, their 3-oxygenated congeners, and octanoic acid have suggested that 1 is biosynthesized via coupling of a C(5) unit with octanoate, rather than via introduction of a C(3) unit at the alpha position of a decanoate derivative. Further feeding study of [2,3-(13)C(2)]decanoic acid concluded that the former route is operating in the biosynthesis of 1.