The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca2+, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin-fragmin specifically binds PIP2 which competes with actin for the Ca(2+)-sensitive site. However, PIP2 cannot dissociate the actin-fragmin complex nor the actin2-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP2 and at high Ca2+ (> microM) concentrations. In the presence of PIP2, actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP2, concomitant with the increase of cytosolic Ca2+, could promote subcortical actin polymerization.