Brefeldin A-inhibited guanine nucleotide-exchange proteins (GEPs) BIG1 and BIG2 activate ADP-ribosylation factor (ARF) GTPases, which are required for vesicular trafficking. Both molecules contain one or more sites for binding protein kinase A, i.e., A kinase-anchoring protein (AKAP) sequences. Elevation of cell cAMP caused PKA-catalyzed phosphorylation and nuclear accumulation of BIG1 but not BIG2. We then asked whether BIG1 phosphorylation altered its GEP activity. Incubation of BIG1 or BIG2 with PKA catalytic subunits and ATP resulted in retardation of their electrophoretic migration, consistent with PKA phosphorylation. Okadaic acid inhibits many protein phosphatases, including protein phosphatase 1 (PP1) and PP2A, that can reverse PKA-catalyzed phosphorylation. Incubation of HepG2 cells with okadaic acid caused concentration-dependent accumulation of presumably phosphorylated BIG1 and BIG2 with decreased mobility, which was increased by subsequent incubation in vitro with specific recombinant phosphatases, PP1γ > PP2A ≫ PP1α. For assays of GEP activity, BIG1 and BIG2 were immunoprecipitated from cells that had been depleted, respectively, of BIG2 and BIG1 by using specific siRNA. GEP activity of each was significantly decreased after incubation with recombinant PKA plus ATP and restored by incubation with PP1γ. In agreement with a role for PP1γ in regulation of BIG, endogenous PP1γ, but not PP1α or β, was immunoprecipitated with BIG1 or BIG2 from microsomal fractions. All observations are consistent with the effects of BIG1 and BIG2 phosphorylation on vesicular trafficking, via alterations in ARF activation and regulatory roles for cAMP, PKA, and PP1γ in ARF activation by BIG1 and BIG2.