The fraction of inward current carried by Ca2+ (FCa 2+) through nicotinic acetylcholine receptors (nAChRs) on acutely isolated rat medial habenula (MHb) neurons was calculated from experiments that simultaneously monitored agonist-induced membrane currents and intracellular [Ca2+], measured with patch-clamp and indo-1 fluorescence, respectively. In physiological concentrations of extracellular Ca2+ (2 mM) at -50 mV, the percentage of current carried by Ca2+ was determined to be roughly 3-4%, which is in close agreement with measurements from other heteromeric nicotinic receptors expressed in peripheral tissue. Among factors that may have affected this measurement, such as Ca2+ influx through voltage-gated Ca2+ channels, the concentration of intracellular Ca2+ buffer, and Ca2+ sequestration and release from intracellular stores, only Ca2+ uptake by mitochondria was shown to confound the analysis. Furthermore, we find that because of the high density of nAChRs on MHb cells, low concentrations of ACh (10 μM) and its hydrolysis product, choline (1 mM), can significantly elevate intracellular Ca2+. Moreover, during persistent activation of nAChRs, the level of intracellular Ca2+ is proportional to its extracellular concentration in the physiological range. Together, these findings support the suggestion that nAChRs may be capable of sensing low concentrations of diffusely released neurotransmitter and, in addition, transfer information about ongoing local synaptic activity by changes in extracellular Ca2+. Copyright © 2007 The American Physiological Society.