It is proposed that the phosphatidylinositol-transfer protein (PI-TP) may function as a carrier of phosphatidylinositol (PI) in the cell. PI-TP occurs in all mammalian tissues examined and appears to be strongly conserved. Its intracellular distribution was studied by immunoblotting and immunofluorescence techniques. PI-TP displays a dual specificity in that it preferentially transfers PI over phosphatidylcholine (PC) between membranes. Its lipid binding site and transfer characteristics were investigated with fluorescent PI and PC analogues containing parinaroyl- and pyrenylacyl-labeled chains. PI-TP is ideally suited for maintaining PI levels in intracellular membranes, possibly the plasma membrane.