The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K d ؍ 10 M) than to the Arp2/3 complex (K d > 200 M). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.Rapid assembly of actin filaments is required for many basic cellular processes including amoeboid motility, endocytosis, and pathogen invasion (1, 2). One important mediator of actin assembly is the Arp2/3 complex, an essential, evolutionarily conserved, seven-subunit protein complex that nucleates and cross-links new actin filaments. The Arp2/3 complex binds the sides of pre-existing actin filaments and nucleates new filaments branching from the old at a characteristic angle of 70°(3, 4). This activity, called dendritic nucleation (4), generates space-filling networks of cross-linked and entangled actin filaments, the growth of which can generate force and power motility (5). In vitro, three factors cooperate to stimulate the nucleation activity of the Arp2/3 complex: a preexisting actin filament, an actin monomer, and a nucleation-promoting factor (NPF) 3 (6, 7). NPFs include members of the WASP/Scar family of proteins (1,8). Several WASP/Scar family proteins are regulated by small GTPases and thus serve to link Arp2/3-dependent actin assembly to upstream signals. The region of WASP/Scar family proteins that binds and activates the Arp2/3 complex is called a VCA domain and is usually located at the C terminus. A canonical VCA domain is composed of three sequences motifs: one or two V (verprolin homology) sequences (also known as WASP homology 2 or WH2 domains) that bind actin monomers; a C (central or connecting) sequence that is essential for nucleation and has been shown to interact with the Arp2/3 complex; and an A (acidi...