The opioid receptor antagonist, naloxone, has been shown to have beneficial effects in the kidney and to be implicated in renal salt and water balance. In the present study the signal transduction pathways utilized by naloxone were studied in an epithelial cell line model of the cortical collecting duct, A6 cells. We found that naloxone has a dual effect depending on the concentration used: at a low concentration (10<sup>–6</sup> M) it antagonized the β-endorphin-dependent increase in cytoplasmic calcium [Ca<sup>2+</sup>]<sub>i</sub>, while at higher concentrations (>10<sup>–5</sup> M) it increased [Ca<sup>2+</sup>]<sub>i</sub> and intracellular inositol phosphate levels. While naloxone-induced increases in [Ca<sup>2+</sup>]<sub>i</sub> occurred in the absence of external calcium, it was significantly stimulated by increasing the external calcium concentration, suggesting that naloxone increases [Ca<sup>2+</sup>]<sub>i</sub> via both calcium release and calcium influx. In polarized A6 cell monolayers naloxone inhibited the activity of the Na<sup>+</sup>/H<sup>+</sup> exchanger (NHE) only when added to the basolateral cell surface. This inhibition of the NHE was prevented by pretreatment of the cells with either the intracellular calcium chelator, BAPTA or with the protein kinase C inhibitor, calphostin C. These findings demonstrate that naloxone induces a rapid increase in intracellular calcium which inhibits the NHE via the calcium-dependent protein kinase C regulatory pathway.