Synaptic connections between the neurons in the red nucleus (RN) and its extrinsic neurons were studied using rat brain-stem slices. Intracellular records were obtained from the RN neurons. Ipsilateral stimuli to areas in the dorsolateral mesencephalic reticular formation (DLMRF) or substantia nigra (SN) elicited monosynaptic hyperpolarizing postsynaptic potentials (PSPs) in about 95% of RN neurons recorded. The hyperpolarizing PSPs could be reversibly blocked by bicuculline, indicating that they were GABAA receptor-mediated-Cl(-)-inhibitory PSPs. The sites of most inhibitory synapses arising from DLMRF and SN are possibly located on the proximal half of the soma-dendritic membrane of RN neurons, according to the analysis of the IPSPs with Rall's model. In addition, tracing dyes were employed to examine the morphological pathways. After rhodamine B, a retrograde tracer, was applied to the RN in brain slices, the cell bodies of a number of neurons in DLMRF and SN were labeled. These labeled neurons were also immunopositive for glutamic acid decarboxylase (GAD) as revealed from double labeling with an anti-GAD antiserum. The anterograde tracer, tetramethylrhodamine dextran, was applied to the DLMRF or SN and taken up by many neurons in the areas. A portion of these cells extended their processes toward and terminated within the RN. Moreover, electron microscopic examination confirmed that the tetramethylrhodamine dextran-decorated synaptic terminals were present in the RN. The results indicate that the rubral neurons receive direct GABAA receptor-mediated inhibitory inputs from neurons in the DLMRF and SN, which may participate in modulation of rubral outputs.