Arl2 is a ∼20 kDa GTPase in the ADP-ribosylation factor (Arf) family within the Ras superfamily with roles in microtubule dynamics that impact the cytoskeleton, cell division, and cytokinesis. Arl2 has been implicated as a regulator of the pathway responsible for formation of properly folded tubulin heterodimers and in adenine nucleotide transport in mitochondria. The identification and characterization of Arl2 binding partners and regulators of Arl2 activities are critical steps in the further dissection of these and likely other Arl2-dependent functions. This chapter describes methods for preparing recombinant Arl2, loading different radiolabeled guanine nucleotides onto the GTPase, identifying high-affinity Arl2 binding proteins, and assaying Arl2 GTPase activating proteins (GAPs). These methods may also prove useful for studies of other Arls or other GTPases. Copyright 2005, Elsevier Inc. All rights reserved.