The aim of this study was to investigate whether nitroxyl (HNO), a redox variant of the radical gasotransmitter nitric oxide (NO) with therapeutically promising properties, affects colonic ion transport. Changes in short-circuit current (Isc) induced by the HNO donor Angeli's salt were recorded in Ussing chambers. Cytosolic Ca2+ concentration was measured with fura-2. The nitroxyl donor induced a concentration-dependent increase in Isc across rat distal colon which was due to a stimulation of chloride secretion. The secretion induced by Angeli's salt (5×10−4 mol/l) was not altered by the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), but was abolished by the HNO scavenger l-cysteine. The response was not dependent on the activity of soluble guanylate cyclase or enteric neurons, but was inhibited by indomethacin. Experiments with apically permeabilized epithelia revealed the activation of basolateral K+ channels and a stimulation of the current carried by the basolateral Na+—K+-pump by Angeli's salt. The secretion induced by Angeli's salt was reduced in the absence of extracellular Ca2+. A prominent increase in the cytosolic Ca2+ concentration was evoked by Angeli's salt predominantly in subepithelial cells within the submucosa, which had the same dependence on extracellular Ca2+ as the Angeli's salt-induced Cl− secretion. Consequently, Angeli's salt induces a soluble guanylate cyclase-independent, Ca2+-dependent Cl− secretion via activation of the Na+—K+-ATPase and of basolateral K+ channels. Cyclooxygenase metabolites produced within the submucosa seem to be involved in this response.