Ammonia, the third respiratory gas in teleost fish, acts as an acute stimulant to ventilation in ammoniotelic rainbow trout. We investigated whether this sensitivity is maintained in trout chronically exposed (1+ months) to high environmental ammonia [HEA, 250 μmol l–1 (NH4)2SO4] in the water, and whether gill neuroepithelial cells (NECs) are involved in ammonia sensing. Hyperventilation was induced both by acute external (NH4)2SO4 exposure [250 or 500 μmol l–1 (NH4)2SO4] and by intra-arterial (NH4)2SO4 injection (580 μmol kg–1 of ammonia) in control trout, but these responses were abolished in chronic HEA animals. Hyperventilation in response to acute ammonia exposure persisted after bilateral removal of each of the four gill arch pairs separately or after combined removal of arches III and IV, but was delayed by removal of gill arch I, and eliminated by combined removal of arches I and II. NECs, identified by immunolabeling against 5-HT, were mainly organized in two lines along the filament epithelium in all four gill arches. In control trout, NECs were slightly smaller but more abundant on arches I and II than on arches III and IV. Chronic HEA exposure reduced the density of the NECs on all four arches, and their size on arches I and II only. Fura-2 fluorescence imaging was used to measure intracellular free calcium ion concentration ([Ca2+]i) responses in single NECs in short-term (24–48 h) culture in vitro. [Ca2+]i was elevated to a comparable extent by perfusion of 30 mmol l–1 KCl and 1 mmol l–1 NH4Cl, and these [Ca2+]i responses presented in two different forms, suggesting that ammonia may be sensed by multiple mechanisms. The [Ca2+]i responses to high ammonia were attenuated in NECs isolated from trout chronically exposed to HEA, especially in ones from gill arch I, but responses to high K+ were unchanged. We conclude that the hyperventilatory response to ammonia is lost after chronic waterborne HEA exposure, and that NECs, especially the ones located in gill arches I and II, are probably ammonia chemoreceptors that participate in ventilatory modulation in trout.