The putative gene encoding acetyl-CoA carboxylase, accA, has been isolated from Aspergillus nidulans. This single-copy gene has an open reading frame (ORF) of 6864 bp and contains two small introns near the 5'-end. A short ORF upstream of the ATG start codon has been identified in this gene by RT-PCR. Based on sequence homology to acetyl-CoA carboxylases from other organisms, putative biotin-, ATP-, HCO3-- and acetyl-CoA- binding sites have been assigned. Northern data and ACC enzyme-activity measurements from A. nidulans suggested that expression of accA was higher in media containing nitrate than ammonia as a sole nitrogen source. Deletion of accA in A. nidulans was unsuccessful. The failure of A. nidulans to grow in the presence of the ACC-specific inhibitor, soraphen A, supplemented with C16-18 fatty acids suggested that ACC is an essential enzyme.