The autonomic nervous system is fundamental for maintaining homeostasis in health and disease. Although we know substantially how central, sensory and motor nerves signal, there is a paucity of data surrounding autonomic nerve signalling at identified individual neuroeffector junctions. Here, we suggest that noradrenaline and ATP, the major neurotransmitters of the sympathetic nervous system, are focally and intermittently (P ~ 0.01) released to act, at points of close contact, at junctions that are functionally analogous to directed synapses. Various techniques will be discussed to show how the release of these neurotransmitters has been advanced. In particular, a unique confocal microscopy Ca²⁺-imaging method to study Ca²⁺ dynamics simultaneously in nerve terminals and the adjacent smooth muscle cells at individual neuroeffector junctions at a resolution previously unachievable will be demonstrated. The development of a high resolution optical recording technique to detect ATP release from single varicosities on the same terminal branch on an impulse-to-impulse basis will also be described. ATP released from varicose nerve terminals, triggers Ca²⁺ influx through P2X₁ receptors, a neuroeffector Ca²⁺ transient, and the frequency of occurrence of these events can be used to measure neurotransmitter release probability from identified varicosities on the same nerve terminal branch¹. The results confirm the intermittent nature of neurotransmitter release in postganglionic sympathetic nerves innervating rodent vas deferens² with action potential evoked probabilities ranging between 0.01 – 0.05 at 0.1 – 2 Hz. Interestingly however, little postjunctional neuronal noradrenergic Ca²⁺ signalling has been detected to date. This new approach complements well-established techniques to study neurotransmitter release and has the additional advantage of providing greater spatial resolution. These studies will permit further important new insights into the mechanisms controlling sympathetic neurotransmitter release and its effects on smooth muscle cells.