PI(4)P directly interacts with the cytosolic domain of yeast Golgi vacuolar H+-ATPase (V-ATPase) a-isoform, Stv1, and the human Golgi a-subunit isoform. Lys-84 of Stv1 is essential for PI(4)P interaction, and localization of Stv1-containing V-ATPases in vivo requires the PI(4)P interaction. We propose that phosphatidylinositol binding exerts organelle-specific control over V-ATPases.