The cytoskeletal protein vinculin, a putative actin -plasma-membrane linker, has been shown by hydrophobic photo-labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. B i d . Chem. 261, 6912-69181. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts were incubated for 2 h with a 3H-labeled photoactivatable fatty acid, 11-(4-[3-(trifluoromethyl)diazirinyl]phenyl)-[2-"H]undecanoic acid. This resulted in biosynthetic incorporation into cellular lipids of a fraction of the fatty acid added. Following photolysis, vinculin was immunoprecipitated from different subcellular fractions using a specific polyclonal anti-vinculin antibody. The protein was recovered from both the cytosolic and the crude membrane fraction. Vinculin from both fractions incorporated label, but the membrane-associated population was at least eight times more strongly photolabeled than the cytosolic protein. Moreover, photolysis increased only labeling of the membrane-bound but not of the cytosolic protein. These results suggest that the direct interaction of vinculin with the hydrophobic core of the phospholipid layer observed in vitro may also be relevant in intact cells, and may be involved in its function as a linker protein.The widely distributed protein vinculin has been suggested to participate in actin-filament -plasma-membrane linkage. This hypothesis is mainly based on the highly specific location of vinculin in areas where this linkage occurs [l]. However, conclusive evidence confirming this hypothesis is still lacking and the molecular mechanism of the proposed linkage of vinculin to both actin filaments and the plasma membrane has not yet been clarified. Recent experiments suggest very complex interactions. Besides vinculin, the proteins talin and a-actinin are also components of areas of plasma-membraneactin attachment, such as for instance focal contacts, areas of closest contact between cultured cells and the substrate. Vinculin has been shown to interact in vitro both with talk [2] and with a-actinin [3]. Only the latter protein has conclusively been shown also to interact with actin [4]. Talin itself binds in vitro to the purified transmembrane fibronectin receptor [5]. Talin, vinculin and a-actinin may therefore constitute a chain of attachment from the plasma membrane to the actin filaments. However, such a complex has not yet been demonstrated to occur in situ. A different mechanism may be operative in adherens junctions, where only vinculin, but not talin, is present [6].Not only proteins, but also lipids may play a role in the interaction of vinculin with membranes. Vinculin has been shown to interact in vitro with acidic phospholipids [7] and, using a photoactivatable phospholipid, to insert into the hydrophobic core of liposome bilayers [8]. This insertion is critically dependent on the presence of acidic phospholipids and occurs at physiological salt and phospholipid concentrations, although it is maximal at low ionic str...