Sympathetic neurons of the rat superior cervical ganglion (SCG) display a chloride conductance that is activated by acetylcholine. Concomitant activation of nicotinic and muscarinic receptors is required for full activation and protein kinase C (PKC) is central to this process. The delayed onset of ICl(m) (5-10 secs) following a rise in intracellular calcium ([Ca2+]i) at physiological temperatures suggests that the activation mechanism of this conductance is quite distinct from other calcium-activated chloride currents previously described. Objective: To characterise the transduction mechanism and the molecular correlates for ICl(m). Methods: In cultured neurons ICl(m) was recorded by perforated-patch while [Ca2+]i was monitored using Fura-2. In intact ganglia, recordings were made with sharp electrodes using the switch-clamp Over-expression of molecular candidates was achieved by intranuclear((technique. microinjection of cDNA plasmids, whilst 'knockdown' was investigated using siRNA. Results: ICl(m) was present in intact nodose and SCG as well as in dissociated neurons suggesting a physiological role for ICl(m). In cultured dorsal root ganglion neurons, the expression of the current developed with time (1-5 days) in all cell types whilst in SCG neurons ICl(m) was found consistently. Knockdown of the A-kinase anchoring protein (AKAP150) attenuated the current, as did disruption of the cytoskeleton. We have considered that members of either the TMEM16 and Bestrophin series might underlie ICl(m). Immunostaining showed the presence of TMEM16A in SCG neurons, and knockdown of the endogenous protein reduced the mean amplitude of ICl(m). Interestingly, over-expression of TMEM16A and Bestrophin 1 also decreased ICl(m). Conclusions: Scaffolding proteins and the cytoskeleton are required for the local signalling processes that lead to activation of ICl(m). The search for the molecular correlate continues, with a member of the TMEM16 series currently the best candidate.