Slices of rabbit hippocampus were preincubated with [3H]serotonin then superfused continuously and stimulated twice electrically. The stimulation-evoked overflow of tritium was Ca2+-dependent, tetrodotoxin-sensitive and subject to modulation by serotonin autoreceptors. It was decreased by various adenosine receptor agonists in an order of potency that was typical for A1-(Ri-) receptors: N6-cyclohexyladenosine greater than (-)N6-phenylisopropyladenosine greater than 5'-N-ethylcarboxamideadenosine greater than (+)N6-phenylisopropyladenosine = adenosine. The effects of the agonists were antagonized by 8-phenyltheophylline. The hypothesis that endogenous adenosine influences hippocampal serotonin release is supported by the following findings: both the adenosine receptor antagonists (theophylline or 8-phenyltheophylline (10 microM, each)) and the enzyme adenosine deaminase (10 micrograms/ml) increased, whereas R-E 244 (3 microM), an inhibitor of adenosine uptake, significantly decreased the evoked tritium overflow. When 8-phenyltheophylline was present throughout superfusion the effects of both R-E 244 and exogenous adenosine deaminase were abolished. It is concluded that serotonin release in the rabbit hippocampus is depressed by endogenous adenosine via A1-(Ri-) receptors.