The fraction of inward current carried by Ca²⁺ (FCa²⁺) 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 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 via changes in extracellular Ca²⁺.