We studied the effects of varying extracellular concentrations of Ca++ and Mg++ on cytosolic Ca++ concentration and PTH release by incorporating the Ca++-sensitive, fluorescent dye "QUIN 2" into dispersed bovine parathyroid cells. Increasing extracellular Ca++ from 0.5 to 2.0 mM led to a dose-dependent increase in cytosolic Ca++ from 179 +/- 8 to 646 +/- 68 nM (mean +/- SE, N = 13; P less than 0.01), which correlated closely (r = -.987, P less than .001) with the suppression of PTH secretion by the same Ca++ concentrations. Raising extracellular Mg++ from 0.5 to 3 mM also caused a dose-related increase in cytosolic Ca++ from 176 +/- 10 to 277 +/- 15 nM (N = 12, P less than 0.01) which correlated (r = -.959, P less than .01) with inhibition of PTH release. These results show a close inverse relationship between cytosolic Ca++ and PTH release with alterations in extracellular Ca++ or Mg++. Cytosolic Ca++ may, therefore, act as a second messenger mediating the effects of these divalent cations on PTH release.