Based on a slice superfusion technique, this study investigated the release of acetylcholine, noradrenaline and serotonin in the hippocampus of aged rats (25-27 months) showing no or severe deficits in a spatial reference-memory task (water maze). Young adults (3-5 months) were used as controls. 3,4-diaminopyridine (3,4-DAP), a potassium channel antagonist which increases neuronal excitability, was used to evoke the overflow of the three neurotransmitters. The release of [3H]noradrenaline induced by stimulation of presynaptic nicotinic receptors was also assessed. The experiment compared the accumulation and 3,4-DAP-evoked (or nicotine-evoked) overflow of [3H] in hippocampal slices preincubated with [3H]choline, [3H]noradrenaline, or [3H]serotonin. In aged rats, only the accumulation of [3H]serotonin was reduced significantly (-17%). In percent of tissue-[3H], the 3,4-DAP-evoked overflow of [3H]serotonin was increased (+28%), and that of [3H]acetylcholine was reduced (-23%) in the aged rats. The nicotine-evoked overflow of [3H]noradrenaline was not altered in aged rats. There was a significant correlation of water-maze performance (distance to platform) and evoked overflow of [3H]serotonin. It is concluded that hippocampal cholinergic functions are more altered by aging than noradrenergic or serotonergic ones. Excessive excitability of serotonergic terminals, perhaps in addition to cholinergic dysfunction, might be a crucial factor accounting for age-related cognitive deficits in the present population of rats.