Adhesion of normal, anchorage-dependent cells to a solid substratum leads to activation of the Na+/H+ antiporter and elevation of intracellular pH. These effects are mediated by extracellular matrix proteins, such as fibronectin, and their receptors, the integrins. Experiments using pharmacological inhibition and down-regulation of protein kinase C (PKC) in C3H 10T1/2 cells show that platelet-derived growth factor induces activation of the Na+/H+ antiporter by means of a PKC-dependent pathway in adherent cells but cannot do so in poorly adherent cells. Poorly adherent cells are, however, able to elevate intracellular pH in response to a phorbol ester, indicating that PKC and subsequent steps in the pathway are functional. These results indicate that coupling of platelet-derived growth factor to PKC activation requires cell adhesion.