Changes in ionized Ca++ concentration are thought to be the main factor in the control of parathyroid hormone (PTH) secretion from the parathyroid cell. In other secretory cells, functional Na+ channels and a Na+-Ca++ exchange have been shown to play a role in the regulation of hormone excretion. To assess the role of Na+-Ca++ exchange on PTH secretion by collagenase-dispersed bovine parathyroid cells, we examined the effects of 1) ouabain and harmaline, specific inhibitors of Na+-K+-ATPase; 2) an absence of K+ in the external medium, a maneuver known to inhibit the Na pump (and hence Na+ extrusion from cells); and 3) Na+-free media. Incubations were carried out at 37 C for 180 min, and under the conditions used, hormone secretion was linear for at least 240 min. The secretion of PTH decreased appropriately at a high (3.0 mM) Ca++ concentration as compared to that when a low Ca++ concentration (0.5 mM) was present in the medium. The mean decrease in PTH secretion at high Ca++ concentrations averaged 40.5 ± 3.2%. Ouabain (2 X 10-4 M) added to cells incubated in 0.5 mM Ca++ medium decreased PTH secretion by 46.5 ± 4.1%. Similar effects were seen with ouabain concentrations as low as 10-7 M. Potassium-free medium decreased PTH secretion compared to that from cells incubated with 5 mM K+ at a low (0.5 mM), but not at a high (2.0ndash;3.0 mM), calcium ion concentration in the medium. This inhibition of hormone secretion in potassium-deficient medium was overcome when the potassium ion concentration was restored to 5 mM. Substitution of Na+ by choline chloride (at 0.5 mM Ca++) suppressed PTH secretion by 27.7 ± 2%, whereas the simultaneous addition of ouabain to the choline medium produced an overall decrease in PTH secretion of 35.2 ± 4%. Harmaline concentrations of 1–20 mM inhibited hormone secretion an average of 46 ± 5% in low calcium medium. None of these experimental conditions decreased hormone secretion significantly below the basal rate of secretion imposed by a high (3.0 mM) medium calcium concentration. Based on these results, we suggest the following. 1) Ouabain and harmaline affect PTH secretion by their action on Na+-K+-ATPase, since inhibition of the Na+ pump by K+ removal yielded similar results. Both of these maneuvers presumably increase intracellular Na+ and decrease the Na+ leak into the cells. 2) A decrease in Na+-Ca++ exchange either by blocking the Na pump or removing external Na+ would decrease the extrusion of Ca++ from the cells, thus increasing intracellular Ca++, which, in turn, will suppress PTH secretion. 3) The restoration of hormone secretion by the readdition of potassium to parathyroid cells incubated in a potassium-free medium suggests that an irreversible toxic effect to the cells does not account for the results observed.