We recently identified three AKAP12 isoforms that are differentially regulated by distinct promoters. During a screen to identify molecular determinants distinguishing the activities of these promoters, we found a potential binding site for the serum response factor (SRF) in the promoter of the ubiquitously expressed AKAP12alpha isoform. SRF is an evolutionarily conserved transcription factor that governs disparate programs of gene expression linked to cellular growth and differentiation. Using a combination of reporter assays and RNA interference, we demonstrate that SRF is required for AKAP12alpha expression. SRF regulates the activity of the AKAP12alpha promoter through two conserved CArG boxes that bind SRF with different affinities. Unlike other SRF-dependent genes, AKAP12alpha is not regulated by growth or differentiation stimuli. Molecular analysis of the AKAP12alpha SRF-binding sites, or CArG boxes, indicates that sequences flanking these sites are the determinants of sensitivity to SRF-activating signals. Specifically, the AKAP12alpha CArG boxes are shielded from growth stimulation by the absence of a binding site for Ets transcription factors. Similarly, sensitivity to the differentiation-associated co-factor, myocardin, was also determined by responsive flanking sequence; however, unlike growth stimuli, sensitivity to myocardin was found to also be dependent on a consensus CArG box. Collectively, our data demonstrate that AKAP12alpha belongs to a novel class of atypical SRF-dependent target genes. Furthermore, we provide new insight into the role of flanking sequences in determining sensitivity to SRF-myocardin activity.