The effect of (+)-tubocurarine (TC) on the release of [3H]acetylcholine from the rat phrenic nerve-hemidiaphragm preincubated with [3H]choline was investigated at different stimulation frequencies and train lengths. At 0.5 Hz (100 pulses) TC failed to modulate the evoked acetylcholine release. A slight (30%) inhibition was observed at 1 Hz (100 pulses). Release of acetylcholine evoked at 5, 25 and 50 Hz (100 pulses) or 100 Hz (200 pulses) was markedly reduced by TC. The degree of inhibition (60%) was similar between 5 Hz and 100 Hz. A concentration of 1 mumol/l TC was a maximal effective concentration at 5 Hz whilst at all higher stimulation frequencies a 10-fold higher concentration was necessary for the maximal effect. When 300 pulses were continuously applied at 5 Hz or 50 Hz TC caused only a slight inhibition (20%). Additionally, the phrenic nerve was stimulated intermittently. Trains of 15 pulses were repeated 10 times with an interval of 3 s between each train. Under this latter stimulation condition TC failed to reduce acetylcholine release. It is concluded that nicotinic autofacilitation of acetylcholine release from the motor nerve operates at frequencies and stimulation conditions similar to the pattern of nerve activity under in vivo conditions. At least more than 15 pulses are required before the nicotinic autofacilitation becomes apparent. It appears unlikely that the TC induced fading of end-organ responses can only be attributed to a blockade of the presynaptic nicotine receptors.