The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [(14)C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.