Actinonin is a naturally occurring hydroxamic acid and a potent inhibitor of the essential cotranslational protein processing enzyme peptide deformylase. Actinonin has both pre- and post-emergence herbicidal activity, but it is rapidly metabolized by plants, thus limiting herbicidal efficacy. Studies designed to elucidate the metabolic fate of actinonin revealed that after absorption actinonin was metabolized by tobacco plants with only about 17% of the parent compound remaining 48 h after application. Subcellular fractionation revealed that a microsomal fraction was capable of metabolizing actinonin in vitro. Two actinonin metabolites were isolated by reverse-phase high-performance liquid chromatography and identified by mass spectrometric analyses. The major metabolite was derived from the hydrolysis of the hydroxamate group to its corresponding acid, and a relatively minor metabolite through reduction of the hydroxamate group to the corresponding amide. Both metabolites were functionally inactive as inhibitors of peptide deformylase. These results provide rationale for the low efficacy of actinonin as a broad-spectrum herbicide, and identify functional groups in actinonin targeted by plants during detoxification. This information may facilitate the design and synthesis of actinonin analogues with increased herbicidal efficacy.