The release of serotonin was studied in superfused rabbit brain cortex slices that had been preincubated with [(3)H]serotonin. The slices were stimulated by single electrical pulses or by trains of 4 pulses delivered at 0.011, 1 or 100 Hz. The overflow of tritium elicited by these stimuli was calcium-dependent and tetrodoxin- and Mg(2+)-sensitive. When the superfusion medium contained nitroquipazine or fluvoxamine, the overflow elicited by 4 pulses at 0.011 or 1 Hz was about 2-fold, whereas the overflow elicited by 4 pulses/100 Hz was about 3.7-fold the single pulse-evoked overflow. Metitepin increased little the overflow of tritium elicited by single pulses or 4 pulses/100 Hz, but increased by up to about 150% the overflow evoked by 4 pulses at 1 or 0.011 Hz. Unlabelled serotonin inhibited the evoked overflow in a similar manner, irrespective of the stimulation conditions. When the superfusion medium did not contain serotonin uptake inhibitors, the overflow elicited by a single pulse was too low to be reliably measured. Metitepin increased only slightly the overflow at 4 pulses/0.011 Hz or 4 pulses/100 Hz but increased by up to about 160% the overflow at 4 pulses/1 Hz. The results indicate that the release of serotonin elicited by single pulses as well as by 4 pulses/100 Hz is subject to only a small tonic, stimulation-independent presynaptic autoinhibition, and under these conditions the three pulses following the first one at intervals of 10 ms release about the same amount of transmitter as does pulse No. 1. In contrast, stimulation-dependent presynaptic autoinhibition develops in trains of 4 pulses delivered at 0.011 Hz (only when serotonin re-uptake is blocked) or 1 Hz so that there is a marked fall in the release elicited by each pulse in the course of the train. The total release elicited by such short, autoinhibited trains is dominated by the large response to pulse No. 1. It seems possible that more presynaptic autoreceptors are available for exogenous agonists than for released serotonin.