During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.