Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca2+ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca2+ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd2+ or EGTA from the external solution. The results are explained by a model with parallel Ca2+ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca2+-activated K+ channels at a distance from the release site.