A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3'-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5'-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 microM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.