The Arf GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors (GEFs) to facilitate GTP loading and GTPase activating proteins (GAPs) to promote GTP hydrolysis. While there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs, are characterized by the presence of at least one N-terminal PH domain and two or more Ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including post-translational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular ArfGTP. In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, while ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated, through a mechanism involving the N-terminal sterile-alpha motif (SAM). AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent upon its N-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, suggesting that regulatory crosstalk exists among Arf isoforms.