The elusive coupling between endoplasmic reticulum (ER) Ca 2 + stores and plasma membrane (PM) ''store-operated'' Ca 2 + entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca 2 + entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca 2 + entry channels to Ca 2 + store depletion. Coupling is concluded to be mediated by a physical ''secretion-like'' mechanism involving close but reversible interactions between the ER and the PM.