Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca# + and guanosine 5h-[γ-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20-30 min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25 µM ATP can extend responsiveness significantly ; this effect is maximal at 50 µM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, rundown is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPα and PITPβ have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be