In response to signaling, the Arp2/3 complex (actin-related proteins 2 and 3 complex) is activated by binding the C-terminal (WA) domain of proteins of the Wiskott-Aldrich Syndrome family to promote the formation of a branched actin filament array, responsible for cell protrusion. The Arp2/3 complex exists in different structural/functional states: the inactive Arp2/3, the activated WA.Arp2/3 complex, the ternary G-actin.WA.Arp2/3 complex, which branches the filaments. This work addresses the role of ATP binding in Arp2/3 function. Using photo-cross-linking, hydrodynamic, and fluorescence techniques, we show that in the inactive Arp2/3 complex only one rapidly exchangeable ATP is tightly bound to Arp3 with an affinity of 10(8) m(-1). Upon activation of the Arp2/3 complex by WA, ATP binds to Arp2 with high affinity (10(7) m(-1)), implying that a large structural change of Arp2 is linked to Arp2/3 activation. ATP is rapidly exchangeable on Arp2 and Arp3 in WA.Arp2/3 and G-actin.WA.Arp2/3 complexes. ATP is not hydrolyzed in inactive Arp2/3, in WA.Arp2/3, nor in G-actin.WA.Arp2/3. Arp2 has a greater specificity than Arp3 for ATP versus ATP analogs. Using functional assays of actin polymerization in branched filaments, we show that binding of ATP to Arp2 is required for filament branching.