The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.