The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated kinase family (Lee, S.-F., Egelhoff, T. T., Mahasneh, A., and Côté, G. P. (1996) J. Biol. Chem. 271, 27044-27048). MIHCK incubated with MgATP in the absence of effectors incorporates 1 mol of phosphate/mol, resulting in an approximately 40-fold increase in kinase activity. Sequence analysis of tryptic peptides has identified the major site of phosphorylation as Ser-8. A peptide and a glutathione S-transferase fusion protein containing the Ser-8 phosphorylation site were good substrates for MIHCK, indicating that MIHCK can catalyze its own activation. Guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-Rac1 stimulates MIHCK autophosphorylation and kinase activity 10-fold. Phosphatidylserine, phosphatidylinositol, and phosphatidylinositol 4,5-bisphosphate, but not phosphatidylcholine or sphingosine, were as effective as GTPgammaS-Rac1 in enhancing MIHCK autophosphorylation and activity. Acidic lipids and GTPgammaS-Rac1 induced the autophosphorylation of a similar set of sites as judged by two-dimensional tryptic peptide maps. It is proposed that GTP-Rac and acidic phospholipids function cooperatively to associate MIHCK with membranes. Ca2+-calmodulin bound MIHCK and inhibited activation by acidic phospholipids but not by GTPgammaS-Rac1. These studies reveal a number of similarities between the regulatory properties of the Dictyostelium and Acanthamoeba MIHCK, suggesting that the signaling pathways that control myosin I are conserved.