The cell cortex of Dictyostelium amebae contains an actin-rich cytoplasmic matrix. Changes in geometry of this matrix are believed to regulate protrusive activity and motility of the cell cortex. Two actin-binding proteins (120,000 and 95,000 daltons [120K and 95K]) are present in the cell cortex, and their properties, many of which are described here for the first time, suggest that they regulate growth and organization of cortical microfilaments. The 120K protein is a flexible dimer 35 nm in length with a native molecular mass of 241,000. It nucleates the polymerization of actin and crosslinks the filaments to form branched networks like those seen in situ in the cell cortex. The production of a branched network of short crosslinked filaments results in a lattice that would theoretically generate the maximum rigidity with minimum amount of polymer. This sort of lattice would be very useful as a space-filling cytoskeleton capable of resisting deformation. The 120K protein inhibits the actin-stimulated Mg ATPase of myosin. Competition for actin binding between 120K and myosin, the impenetrability of the 120K-actin network to myosin, and the rigidity of actin filaments that are crosslinked by 120K could all contribute to the decrease in the actin-stimulated Mg ATPase of myosin. The properties of 120K are consistent with a role for this protein in regulating the site of actin filament growth and gelation in the cell but not the assembly of actin-containing structures that would participate in force generation by a sliding-filament mechanism involving myosin. The 95K protein is a rigid dimer 40 nm in length with a native molecular mass of between 190,000 and 210,000. Its physical and antigenic properties lead us to conclude that the 95K protein is Dictyostelium alpha-actinin. Unlike 120K, it crosslinks actin filaments into lateral arrays and increases the actin-stimulated Mg ATPase of myosin. Both activities are regulated by Ca2+. The properties of 95K are consistent with a role in organizing actin filaments in the cell into lateral arrays that are capable of efficient interaction with myosin to produce force for cell motility.