High-resolution imaging of autonomic terminals in living organs is limited by the difficulty of determining the nerve terminal type on the microscope stage. One potentially useful tool for identifying sympathetic terminals is bretylium. By measuring the nerve terminal impulse, bretylium has been shown to abolish nerve terminal action potentials in the guinea pig vas deferens (Brock & Cunnane, 1988), but in the mouse, most nerve terminal Ca²⁺ transients are not abolished by bretylium (Jackson & Cunnane, 2002). To further characterize the action of bretylium in the mouse vas deferens, its actions on nerve terminal Ca²⁺ kinetics and neuroeffector Ca²⁺ transients (NCTs; focal smooth muscle Ca²⁺ transients rapidly triggered by local ATP release) were determined. Formaldehyde-glutaraldehyde-induced catecholamine fluorescence (FAGLU) was used in conjunction with orthograde filling of the Ca²⁺ indicator Oregon Green 488 BAPTA-1 10 kDa dextran (Brain & Bennett, 1997) to identify sympathetic terminals on the stage of a UV confocal microscope. 113 of 117 nerve terminals filled with the Ca²⁺ indicator were FAGLU positive (93 – 99%; n = 7 preparations) and are hence argued to be sympathetic. Bretylium did not abolish the field-stimulus-induced Ca²⁺ transient in any of the terminals tested in this series (n = 7). However, bretylium did cause both an increase in the delay between stimulation and the arrival of the action potential in the terminal (by 2 ± 0.4 ms) and the absolute refractory period to paired-pulse stimuli (by 4 ± 2 ms). In other experiments, smooth muscle cells were filled with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM. Bretylium (10 µM) abolished NCTs at 60/61 junctions over the course of 2 hours. This suggests that sympathetic terminals can be identified by the presence of NCTs in smooth muscle cells adjacent to nerve terminal varicosities. Given that the vast majority of terminals loaded with the Ca²⁺ indicator using this protocol contain catecholamines (positive UV fluorescence after FAGLU) these findings suggests that in addition to its established action on nerve terminal action potential propagation, bretylium abolishes neurotransmitter release downstream of Ca²⁺ influx. Measurement of the bretylium-induced changes in absolute refractory period permits a simple method by which living sympathetic terminals can be identified.