Using an anti-NHE1 antibody, we demonstrate the presence of a Na⁺/H⁺ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1_basal), at the junction between inner and outer cells layers (NHE1_inter) and along the lateral membranes (NHE1_later) of all cells of the duct. At the luminal membrane, no staining was demonstrated neither for NHE1 nor NHE3. To investigate Na⁺/H⁺ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO₃^--free conditions. In the presence of basolateral EIPA, an acidification of 0.29 ± 0.03 pH units was observed while no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 ± 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinisation, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1_inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in pH_i regulation.