1. We made whole-cell recordings from CA1 pyramidal cells in the rat hippocampal slice preparation to study the calcium (Ca2+) dependence of depolarization-induced suppression of inhibition (DSI). DSI is a retrograde signalling process in which voltage-dependent Ca2+ influx into a pyramidal cell leads to a transient decrease in the release of GABA from interneurons. 2. To investigate the Ca2+ dependence of DSI without altering extracellular divalent cations, we varied the type and amount of Ca2+ chelator (EGTA or BAPTA) in the recording pipette (keeping the chelator : Ca2+ ratio constant). Evoked inhibitory postsynaptic currents (IPSCs) were induced in the presence of antagonists of ionotropic glutamate receptors. DSI was induced by depolarizing voltage steps, lasting from 0.025 to 5 s, to 0 mV. 3. DSI was directly dependent on the duration of the voltage step used to induce it, from threshold up to a maximal value of IPSC suppression, whether EGTA or BAPTA was used, and whether their concentrations were 0.1, 0.5 or 2 mM. For instance, a voltage step lasting 1.37 s produced half-maximal DSI with 2 mM BAPTA, but with 0. 1 mM BAPTA, half-maximal DSI was achieved with a step lasting 0.186 s. Peak DSI was the same in all cases, and DSI was blocked with either 10 mM EGTA or BAPTA in the pipette. Bath application of carbachol could overcome the block of DSI by 10 mM EGTA but not by 10 mM BAPTA. 4. We calculated that a voltage step lasting approximately 100 ms would be necessary to activate half-maximal DSI in the absence of exogenous Ca2+ buffers. 5. Log-log plots of calculated total Ca2+ influx, estimated from time integrals of Ca2+ currents, versus DSI yielded a straight line with a slope of approximately 1, and increasing extracellular [Ca2+] from 2.5 to 5 mM did not change the slope. 6. The time course of decay of DSI was well described by an exponential function with a time constant of approximately 20 s and was not affected by changes in either concentration or type of Ca2+ buffer. 7. The data suggest that, in its Ca2+ dependence, DSI more closely resembles the slow release of neuropeptides and hormones than it does the process of fast release of many neurotransmitters.