Neuroblastoma N18TG-2 cells cannot synthesize or release acetylcholine (ACh), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG-2 cells with cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediatophore 16-kDa subunit, or both. Cells transfected only with ChAT synthesized but did not release ACh. Cells transfected only with mediatophore expressed Ca2+-dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatophore cDNAs released the ACh that was endogenously synthesized. Synaptic-like vesicles were found neither in native N18TG-2 cells nor in ChAT-mediatophore cotransfected clones, where all the ACh content was apparently cytosolic. Furthermore, restoration of release did not result from enhanced ACh accumulation in intracellular organelles consecutive to enhanced acidification by V-ATPase, as Torpedo 16 kDa transfection did not increase, but decreased the V-ATPase-driven proton transport. Using ACh-sensitive Xenopus myocytes for real-time recording of evoked release, we found that cotransfected cells released ACh in a quantal manner. We compared the quanta produced by ChAT-mediatophore cotransfected clones to those produced by clones transfected with mediatophore alone (artificially filled with ACh). The time characteristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proportion of their initial ACh store. Hence, expression of mediatophore at the plasma membrane seems to be necessary for quantal ACh release; the process works more efficiently when ChAT is operating as well, suggesting a functional coupling between ACh synthesis and release.