alpha-Actinin has been proposed to be the actin-plasma membrane linker. This assumption is based on the discovery of direct interaction of alpha-actinin with two specific lipids, diacylglycerol and palmitic acid [Burn, P. (1988) Trends Biochem. Sci. 13, 79-83]. In our study, the binding of alpha-actinin with vesicles containing negatively charged phospholipids was measured by the method of 90 degrees light-scattering. Our results show that alpha-actinin is able to bind membranes containing negatively charged phospholipids, but not to bind membranes composed of neutral lipids only. Diacylglycerol and palmitic acid, on the other hand, have little effect on the binding of alpha-actinin to lipid vesicles. Analysis of binding isotherms in terms of a membrane binding model gave apparent dissociation constants which varied between 0.2 and 3 microM over a range of 5-20 mol % negatively charged phospholipid. Comparing the kinetics of alpha-chymotrypsin digestion of alpha-actinin in solution to those of vesicle-bound alpha-actinin, it can be seen that the cleavage site at the junction between the C-terminal and the central rod domain of alpha-actinin and another cleavage site on the C-terminal domain can be most effectively protected by its membrane binding. Analysis of the amide I and II regions of Fourier-transform infrared spectra of alpha-actinin revealed that the association of alpha-actinin with negatively charged phospholipid vesicles resulted in some perturbation of the protein secondary structure. Monolayers containing negatively charged phospholipid were layered and incubated on the surface of a polymerization solution of actin and alpha-actinin, and observed with an electron microscope. The results show that the bundle structure of actin filaments can be formed if diacylglycerol and palmitic acid are present in lipid layers.