Abstract We have previously developed a low-volume perfusion-superfusion system for studying neurotransmitter release in small blood vessels. We now extend this technique to allow for the measurement of neurotransmitter release with short stimulation trains in order to examine modulating factors such as activation of prejunctional receptors or the effects of altered external calcium. Segments of rat tail artery were perfused in the presence of deoxycorticosterone and cocaine (10 −5 M). For short trains, nerves were activated with five sequential trains, each 4 sec in length (total of 160 pulses) at 8 Hz. For long trains, nerves were activated continuously for 3 min for a total of 1440 pulses. Norepinephrine in the perfusate and tissue norepinephrine were quantitated with high-performance liquid chromatography (HPLC) and electrochemical detection. Fractional norepinephrine release was significantly greater with short as compared to long trains when extracellular calcium was maintained at 1.6 mM. When nerves were stimulated with short trains, fractional norepinephrine release was very sensitive to altered extracellular calcium, with a significant decline when extracellular calcium was reduced to 1 mM and a significant increase when extracellular calcium was increased to 5 mM. In contrast, with long-stimulation trains increases or decreases in calcium had no effect on fractional norepinephrine release. This unique method facilitates the study of norepinephrine release with short trains or low-frequency activation and will also make it easier to study presynaptic receptor function as well as interactions between extracellular calcium and presynaptic effectiveness.