The myristoylated, alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells, and has been implicated in diverse cellular processes including neurosecretion, fibroblast mitogenesis, and macrophage activation. In macrophages that have spread on the substratum, MARCKS has a punctate distribution at the cell-substratum interface of pseudopodia and filopodia. At these points, MARCKS co-localizes with vinculin and talin. Activation of PKC with phorbol esters results in the rapid disappearance of punctate staining of MARCKS, but not vinculin or talin, and is accompanied by cell spreading and loss of filopodia. The morphological changes and disappearance of punctate staining follow a time-course that closely approximates both the PKC-dependent phosphorylation of MARCKS, and its phosphorylation-dependent release from the plasma membrane. Our results suggest a role for PKC-dependent phosphorylation of MARCKS in the regulation of the membrane cytoskeleton.