A single stimulus applied to sympathetic neurons co-cultured with cardiac cells produced a very small increase in the release of tritiated norepinephrine and intracellular free Ca2+ concentration ([Ca2+]i), but evoked a typical neuronal action potential. Treatment with 10 mM tetraethylammonium caused a dramatic increase in the responses to a single stimulus, and [3H]norepinephrine release remained above baseline for as long as 20 s. [Ca2+]i increased in cell bodies and neurites from basal levels of 50 to 100 nM to over 300 nM. [Ca2+]i remained elevated for 18 +/- 1 s and required 6.8 +/- 0.5 s for 50% recovery to basal levels. Action potential duration at 50% repolarization was increased from 4.5 +/- 0.3 ms to 88 +/- 20 ms and resting membrane potential decreased from -55 +/- 2 to -43 +/- 4 mV in the presence of tetraethylammonium. Repetitive firing was not observed after a single stimulus in current clamped neurons before or during tetraethylammonium exposure. These findings show a direct relation between action potential duration, Ca2+ entry and transmitter release in response to a single stimulus.