The median preoptic nucleus (MnPO) holds a strategic position in the hypothalamus. It is adjacent to the third ventricle; hence, it can directly access the ionic composition of the CSF. MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as central sensors of extracellular Na(+) concentration ([Na(+)](ext)). The mechanism underlying Na(+) sensing involves the atypical Na(+) channel, Na(X). Here we sought to determine whether Na(+) influx in Na(+) sensors is actively regulated via interaction with other membrane proteins involved in cellular Na(+) homeostasis, such as Na(+)/K(+)-ATPase. The Na(+)/K(+)-ATPase role was investigated using patch-clamp recordings in rat MnPO dissociated neurons. Na(+) current evoked with hypernatriuric solution was diminished in the absence of ATP/GTP, indicating that Na(+)/K(+)-ATPase play a central role in [Na(+)](ext) detection. Specific blockers of α1 and α3 isoforms of Na(+)/K(+)-ATPase, ouabain or strophanthidin, inhibited this Na(+) current. However, strophanthidin, which selectively blocks the α1 isoform, was more effective in blocking Na(+) current, suggesting that the Na(+)/K(+)-ATPase-α1 isoform is specifically involved in [Na(+)](ext) detection. Although strophanthidin did not alter either the membrane resistance or the Na(+) reversal potential, the conductance and the permeability of the Na(X) channel decreased significantly. Our results suggest that Na(+)/K(+)-ATPase interacts with the Na(X) channel and regulates the high [Na(+)](ext)-evoked Na(+) current via influencing the Na(+) influx rate. This study describes a novel intracellular regulatory pathway of [Na(+)](ext) detection in MnPO neurons. The α1 isoform of Na(+)/K(+)-ATPase acts as a direct regulatory partner of the Na(X) channel and influences Na(+) influx via controlling the Na(+) permeability of the channel.