Feedback inhibitory circuits were characterized electrophysiologically in the CA3 region of organotypic rat hippocampal cultures. Pyramidal cells were impaled with sharp microelectrodes and brief depolarizing current pulses were injected intracellularly to elicit single action potentials. An inhibitory postsynaptic potential (I PSP) was observed at fixed latency after the action potential in 27% of impaled cells (n= 131). These IPSPs were fully blocked by bicuculline, indicating that they were mediated solely by γ-aminobutyric acid type A (GABAA) receptors. They were also blocked by 6-cyano-7-nitro-quinoxaline-2, 3-dione but not d-2-amino-5- phosphonovalerate, indicating that non-N-methyl-d-asparttate receptors were necessary and sufficient for activating interposed GABAergic interneurons. Adenosine (0.1-5 μM) increased the percentage of action potentials that were not followed by IPSPs by reducing the probability of glutamatergic activation of the interneurons. In 18 of 21 experiments adenosine also decreased the mean amplitude of successfully elicited IPSPs, indicating that more than one interneuron participated in the feedback inhibition of those pyramidal cells. In three experiments the non-failure IPSP amplitude was not affected by adenosine, suggesting that only one interneuron participated. Repetitive stimulation at 2-4 Hz decreased the amplitude of non-failure feedback IPSPs and usually increased the number of failures of transmission. These effects were transient and insensitive to the GABAB antagonist CGP 35 348. We conclude that both the excitation of interneurons and the release of GABA from interneurons are modulated by repetitive stimulation.