ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.A DP-ribosylation factors (ARFs) are members of the Ras superfamily of Ϸ20-kDa GTPases that have diverse roles in intracellular vesicular trafficking. ARF binding to donor membranes initiates the formation of vesicles for regulated translocation of protein and lipid molecules among eukaryotic cell organelles. This action requires cycling between inactive, largely cytosolic ARF-GDP, and GTP-bound active, membraneassociated forms (1-3). Conversion of ARF-GDP to ARF-GTP is catalyzed by guanine nucleotide-exchange proteins (GEPs), which accelerate the release of bound GDP to permit GTP binding. All known GEPs, although they differ in molecular size and structure, share a highly conserved central sequence of Ϸ200 aa, the Sec7 domain, that is responsible for this function (4-5). Sensitivity of GEP activity to inhibition by brefeldin A (BFA), a fungal fatty acid metabolite that blocks protein secretion (6), distinguishes 2 major groups. BFA-inhibited GEP (BIG)1 (Ϸ200 kDa) and BIG2 (Ϸ190 kDa) were first purified together in Ͼ670-kDa multiprotein complexes from bovine brain cytosol, based on assays of BFA-inhibited ARF activation (7). The molecules are 74% identical in overall amino acid sequences, with 90% identity in Sec7 and other domains (8). Systematic yeast 2-hybrid screening was used to identify their potentially functional interaction...