Brush border myosin-I (BBMI) is associated with the membrane of intestinal epithelial cells where it probably plays a structural role. BBMI also has been identified on Golgi-derived vesicles in intestinal epithelial cells where it may translocate vesicles into the brush border. However, the mechanochemical activity of BBMI bound to a phospholipid membrane has not been described. This study reports that phospholipid membrane-associated BBMI displays ATPase activity when bound to phospholipids, but does not move actin filaments when associated with a phospholipid bilayer. BBMI does not bind significantly to brush border membrane lipids, which contain about 16% phosphatidylserine (PS), in either a pelleting or planar membrane assay. Similarly, planar membranes containing 20% PS do not bind a significant amount of BBMI. Increasing the concentration of PS to 40% does result in the binding of BBMI to both vesicles and planar membranes. This binding is enhanced with increased Ca2+ concentrations. BBMI retains its ATPase activity when bound to phospholipid vesicles containing 40% PS. However, BBMI attached to a phospholipid bilayer surface does not move actin filaments, even though the amount of BBMI bound to the lipid surface, as reflected by the number of actin filaments associated with bilayer-bound BBMI, is sufficient to observe motility in control experiments. When membrane fluidity is reduced by adding cholesterol to the membrane lipids containing 40% PS, BBMI still binds to the membrane, but again no actin filament motility is observed. The lack of binding by BBMI to brush border membrane lipids and the absence of membrane-associated BBMI mechanical activity suggest that factors in addition to membrane lipids are necessary for membrane-associated myosin-I motility.