1. The effects of depletion of endogenous noradrenaline by reserpine-pretreatment on [(3)H]-noradrenaline overflow elicited by nerve stimulation were determined in the isolated nerve-muscle preparation of the cat's nictitating membrane.2. Reserpine pretreatment (0.3 mg/kg, s.c., 4 days prior to the experiment) reduced the noradrenaline levels in the smooth muscle of the nictitating membrane to about 10% of the control values while granular retention of [(3)H]-noradrenaline had recovered to nearly 40% of the controls.3. In the reserpine-pretreated tissue the fraction release per shock induced by nerve stimulation was 2.2-fold higher than the value obtained in the untreated tissues. This effect was correlated with the degree of depletion of the noradrenaline stores rather than with the decrease in the response of the effector organ.4. Phenoxybenzamine, 2.9 muM reduced the responses to nerve stimulation to the same extent in control and in reserpine-pretreated tissues. Yet, this concentration of phenoxybenzamine increased by 13-fold the overflow of the labelled transmitter in the controls and only by 3-fold in reserpine-pretreated tissues.5. The decrease in effectiveness of phenoxybenzamine in enhancing transmitter overflow after reserpine-pretreatment appears to be due to the decrease in the total release of the transmitter.6. The results obtained support the view that in reserpine-pretreated tissues decreased transmitter output reduces the activation of the presynaptic alpha-adrenoceptors which mediate the negative feed-back mechanism that regulates transmitter release by nerve stimulation.