ADP-ribosylation factor 1 (ARF1) is a 21 kDa GTP-binding protein that regulates multiple steps in membrane traffic. Here, two ARF1 GTPase-activating proteins (GAPs) from rat liver were resolved. The GAPs were antigenically distinct. One reacted with a polyclonal antibody raised against the GAP catalytic peptide previously purified by Makler et al. [Makler, Cukierman, Rotman, Admon and Cassel (1995) J. Biol. Chem. 270, 5232–5237], and here is referred to as GAP1. The other GAP (GAP2) did not react with the antibody. These GAPs differed in phospholipid dependencies. GAP1 was activated 3–7-fold by the acid phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) and phosphatidylserine (PS). In contrast, GAP2 was stimulated 20–40-fold by PIP2. PA and PS had no effect by themselves but PA increased GAP2 activity in the presence of PIP2. The GAPs were otherwise similar in activity. In the presence of phosphoinositides, the Km of GAP1 for ARF1–GTP was estimated to be 8.1±1.6 μM and the dissociation constant for ARF1–guanosine 5′,3-O-(thio)triphosphate (GTP[S]) was 7.4±2.2 μM. GAP2 was similar with a Km for ARF1–GTP of 5.4±1.2 μM and a dissociation constant for ARF1–GTP[S] of 4.8±0.3 μM. Similarly, no differences were found in substrate preferences. Both GAP1 and GAP2 used ARF1 and ARF5 as substrates but not ARF6 or ARF-like protein-2. The potential role of multiple ARF GAPs in the independent regulation of ARF at specific steps in membrane traffic is discussed.