Abstract: Regulated actin polymerization drives many crucial cellular processes including cell motility and endocytosis. An important player in these events is the Arp2/3 complex, which nucleates new actin filaments and organizes them into branched arrays. Arp2/3 function is regulated by a class of proteins known as nucleation promoting factors (NPFs) that bind and activate the complex in response to upstream signaling events. One key regulator of Arp2/3 complex activity is the nucleation promoting factor WASP. While numerous WASP binding proteins have been identified, the native state of WASP in the cell has not yet been fully characterized. To investigate whether WASP is present in cells as part of a macromolecular complex, we analyzed endogenous WASP from Jurkat cells using sucrose gradient centrifugation, and found that it exists primarily within one or more complexes of approximately 90–140 kD. Purification of tagged WASP by tandem affinity purification revealed that roughly 95% of purified WASP was stably associated with the protein WIP. To examine the effect of WIP binding on WASP nucleation promoting activity, we purified recombinant WASP-WIP complex and tested its activation by the WASP activator Nck. While WASP alone and WASP-WIP complex display similar levels of activity in the absence of activating factors, in the presence of Nck, WASP-WIP complex is hyperactivated relative to WASP alone. These experiments suggest that WIP binding to WASP may introduce an additional level of responsiveness to upstream signals. To further examine the molecular interactions involved in Arp2/3 complex binding and activation by NPFs, we used a two-pronged mutagenesis strategy to identify NPF binding sites within the Arp2/3 complex as well as NPF residues required to promote Arp2/3 complex activation. Our experiments identified two conserved, surface-exposed sites in the Arp2/3 complex that are critical for its nucleation activity. One of the sites, a basic region in Arp3, is required for binding to the NPF cortactin, suggesting that it interacts specifically with the acidic domain conserved in all known NPFs. A second site in Arp2 consisting of two conserved isoleucine residues is not required for binding to either the class I NPF WASP, or to the class II NPF cortactin, but has been hypothesized to bind 2 the C region of class I NPFs. To further investigate the role of the WASP C region in Arp2/3 complex activation, we mutated a series of C region residues, and tested the ability of these mutants to promote an activating conformational change in the Arp2/3 complex using fluorescence resonance energy transfer (FRET). Our results show that C region residues that are essential for WASP's NPF activity are required to induce a conformational change in the Arp2/3 complex. Taken together, these results suggest a model in which the A region of WASP family NPFs mediates binding to the Arp2/3 complex via an acidic patch in Arp3, while adjacent C region residues promote a conformational change in the complex, perhaps via interactions with Arp2.