Intracellular recordings were made from presumed dopamine-containing neurons in slices cut from the midbrain of the rat. Focal electrical stimulation produced a hyperpolarizing synaptic potential that was reduced by 75–95% by the GABAB-receptor antagonist 2-hydroxysaclofen (300 microM). 5-HT (3–100 microM) reduced the amplitude of the GABAB synaptic potential by 20–74%, with a 50% reduction at 10 microM, but did not reduce the amplitude of synaptic potentials mediated by GABAA receptors. 5-HT acted presynaptically because hyperpolarizations produced by exogenously administered GABA (1 mM) in picrotoxin (100 microM) were not affected by 5-HT (30 microM). (+/-)-Cyanopindolol (100 nM), a 5-HT1B antagonist, blocked the effect of 5-HT (10 microM); spiperone (1 microM), which is an antagonist at 5-HT1A and 5-HT2 receptors, had no effect. The amplitude of the GABAB synaptic potential was reduced by the 5-HT1B receptor agonists 1-[3-(trifluoromethyl)- phenyl]-piperazine (300 nM) and 7-trifluoromethyl-4-(4-methyl-1- piperazinyl)-pyrrolo[1,2-a]quinoxaline (1 microM), but not by the 5- HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (1 microM) or the 5- HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (10 microM). We conclude that 5-HT activates presynaptic 5-HT1B receptors that inhibit the release of GABA onto GABAB but not GABAA receptors.