The intact ARP2/3 complex is first purified from Acanthamoeba castellanii based on its affinity for the actin-binding protein profilin, and is shown to consist of a stable assembly of seven polypeptides. Two of the subunits are actin-related proteins of the ARP2 and ARP3 subfamilies, giving the complex its name.

The ARP2/3 complex possesses little biochemical activity on its own. However, when engaged by nucleation-promoting factor (NPF) proteins, it is activated to initiate the formation of a new (daughter) filament that emerges from an existing (mother) filament in a y-branch configuration with a regular 70° branch angle. This coupling of nucleation and branching by the ARP2/3 complex is referred to as autocatalytic branching or dendritic nucleation, and is central to its functions in vivo.

Polymerization of actin filaments directed by the Arp2/3 complex supports many types of cellular movements.