We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail. Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background. The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes. We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA. We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.