Epithelial Na⁺ channels play a critical role in the regulation of extracellular fluid volume and the thickness of the fluid layers that cover the mucosal surfaces of the respiratory tract and colon. The intracellular mechanisms that control their activity include inhibitory feedback by the intracellular concentrations of Na⁺ and Cl^–, as well as protein kinases and phosphatases. Feedback regulation by intracellular Na⁺ of the channels is mediated by the ubiquitin protein ligases, Nedd4 and Nedd4-2, which bind to proline-rich motifs (so-called PY motifs) in the carboxy termini of the beta and gamma subunits of the channel. The mechanism by which intracellular Cl^– regulates the channels remains unclear. The kinases that regulate the channels include the G protein-coupled receptor kinase, Grk2, which activates the channels by phosphorylating them and rendering them refractory to the action of Nedd4 and Nedd4-2. Other kinases, such as the serum- and glucocorticoid-induced kinase, Sgk, activate the channels by mechanisms that do not affect Na⁺ feedback regulation of the channels, and may not involve direct phosphorylation of the channel protein. Among the phosphatases that regulate the channels is protein phosphatase I, which counteracts the effects of Grk2 so as to increase the sensitivity of the channels to Nedd4 and Nedd4-2. Epithelial Na⁺ channels are also regulated by a wide variety of extracellular factors. These include hormones such as aldosterone, autocrine factors such as ATP and pathogens such as influenza virus and parainfluenza virus. The intracellular mechanisms by which these agents influence Na⁺ channel activity are known in part. Aldosterone, for example, exerts its immediate actions by increasing expression of Sgk, while the effects of influenza virus are mediated by protein kinase C. The mechanisms by which ATP inhibits the channels are of particular interest since purinergic inhibition of epithelial Na⁺ channels is of potential importance in the treatment of cystic fibrosis and because the inhibitory effects of parainfluenza virus on the channels are mediated by the autocrine action of ATP. ATP is known to act on purinergic receptors in both the the apical and basolateral membranes of epithelia and has been proposed to inhibit epithelial Na channels via increased intracellular Ca²⁺, via protein kinase C, and via changes in the content of phosphoinositides in the cell membrane. We have been using mouse mandibular salivary duct cells studied in the whole-cell patch-clamp configuration and Fischer Rat Thyroid cells transfected with the α-, β- and γ-subunits of mouse ENaC and cultured on permeable supports to screen for novel intracellular regulators of the channels, and to identify the intracellular signalling pathways by which purinergic receptors regulate epithelial Na⁺ channels. Our experiments have ruled out roles for protein kinase C and membrane phosphoinositides in mediating the actions of purinergic receptors on the channels, while indicating that phospholipase Cβ plays only a limited role. They also suggest that the role of intracellular Ca²⁺ in the purineric effects on the channels is mediated by changes in intracellular Cl^–.