Parathyroid hormone (PTH) secretion was investigated in intact cells isolated in vitro. Parathyroid cells from bovine parathyroid glands were obtained through tissue dispersion and cell purification through isotonic Percoll gradients, a newly developed protocol enhancing cell homogeneity and viability. Isolated cells maintained both metabolic viability and plasma membrane intactness for over 3 h at 37 degrees C, as shown by the large ATP/ADP ratios and the high intracellular K+ content (ouabain-sensitive) measured. The rate of PTH secretion was inversely related to the Ca2+ concentrations in the medium; secretion was 54 and 18 ng PTH/mg of protein/min when free Ca2+ in the buffer was 0.8 and 2 mM, respectively. At either Ca2+ concentration, PTH secretion was strongly dependent on cell metabolism; it was inhibited by 80-85% within 10 min when cells were suspended in glucose-free buffer containing either cyanide or oxidative phosphorylation uncoupler. Under these conditions, both cellular ATP production and calcium-dependent PTH release could be partially restored by addition of 5 mM glucose. La3+, Mn2+, Sr2+, Ba2+, and Mg2+ were each tested in a range of 0.5-2.5 mM for their effects in suppressing low calcium-stimulated secretion. La3+ and Mn2+ were about twice as effective as Ca2+ on a molar basis, Sr2+ was similar to Ca2+, Mg2+ was about half as effective, and Ba2+ had almost no effect. These results suggest that the Ca2+-dependent stimulus-secretion coupling of these cells is largely different from that established in other secretory cells and provide an in vitro system to further investigate the regulation of PTH secretion.