Acetyl-Coenzyme A Carboxylase (acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) (ACC), is the biotin containing enzyme that catalyzes the rate-limiting step in the biogenesis of long-chain fatty acids. The regulatory mechanism of acetyl-CoA carboxylase at the gene level has been further established in the present studies. Under stimulated lipogenic conditions, ACC gene is activated and the forms of mRNA with different 5\sp\prime untranslated regions accumulate. This accumulation is the result of an increase in the rate of transcription. Multiple forms of ACC transcripts are generated as a result of differential splicing of two primary transcripts under the influence of two promoters, promoter I and promoter II. In 30A5 cells, ACC activity and mRNA levels increase during the differentiation process. The species of ACC mRNA accumulated are those transcribed from promoter II and there is little or no mRNA from promoter I. The conversion of 30A5 preadipocytes to adipocytes was much more accelerated by the cAMP pretreatment scheme or by the nutrient limitation scheme than by the classical procedure by 4 days. By examining chloramphenicol acetyltransferase (CAT) activities of stably transfected clones containing chimeric constructs between different fragments of the ACC promoter II and the promoterless CAT gene, we established that insulin induction on ACC gene expression and concomitant differentiation of 30A5 preadipocytes to adipocytes requires prior cAMP action and the presence of a specific promoter region which may contain cAMP responsive element(s). From DNase I footprinting analyses and gel retardation assays, we determined that the cis-element for cAMP action and binding of transcription factor(s) in 30A5 nuclear extracts is the AP-2 binding site (β108 to β100), GGGGCTGGG, in the ACC promoter II which also can be recognized by the purified human AP-2 protein