We have previously found that high extracellular calcium (Ca⁺⁺) concentrations inhibit PTH release in association with a threefold to fourfold rise in cytosolic Ca⁺⁺ concentration. Recent data have also shown that low extracellular potassium (K⁺) concentration or ouabain also inhibits PTH release to an extent comparable to that seen with high Ca⁺⁺ and produce a marked rise in the intracellular sodium (Na⁺) content. These results suggested that low K⁺ and ouabain might modulate PTH release through increases in cytosolic Ca⁺⁺ related to alterations in Na⁺-Ca⁺⁺-exchange. In the present studies, we have examined further the mechanism(s) by which inhibition of the Na⁺-K⁺-ATPase regulates PTH release. Exposure of cells loaded with the Ca⁺⁺-sensitive dye QUIN-2 to low K⁺ produced a 10% to 17% increase in cytosolic Ca⁺⁺ at 0.5 to 1.0 mmol/L extracellular Ca⁺⁺, which was statistically significant only at 0.75 mmol/L Ca⁺⁺. In contrast, low K⁺ caused a statistically significant decrease in cytosolic Ca⁺⁺ at 1.5 to 2 mmol/L Ca⁺⁺, while ouabain lowered cytosolic Ca⁺⁺ significantly by 23% to 46% at all Ca⁺⁺ concentrations examined (0.5 to 2 mmol/L). Low K⁺ or ouabain had no effect on cellular levels of ATP or GTP or intracellular pH measured using the pH-sensitive dye BCECF [2′, 7′-bis (carboxyethyl)-5, 6-carboxyfluorescein]. The inhibition of secretion by low K⁺ or ouabain, unlike that due to high extracellular Ca⁺⁺, was not reversed by TPA (12-0-tetradecanoyl phorbol 13-acetate), an activator of protein kinase C. Low K⁺ did produce a modest (30% to 40%) lowering of agonist-stimulated but not basal cAMP content. Thus, changes in Na⁺-Ca⁺⁺-exchange and increases in cytosolic Ca⁺⁺ apparently play no role in the inhibition of PTH release by low K⁺ or ouabain. We also found no evidence that these factors regulate secretion via changes in intracellular pH or protein kinase C activity. The inhibition of PTH release by agents inhibiting the Na⁺-K⁺-ATPase may be related to direct effects of changes in intracellular monovalent cations or to as yet undefined mechanisms.