Measurements of the concentration of intracellular free calcium [( Ca2+]i) were used to screen for the presence of Ca2+-mobilizing receptors on dissociated and purified bovine parathyroid cells loaded with fura-2. Among a wide variety of agents known to mobilize cellular Ca2+ in other cells, only ATP and certain other nucleotides were capable of altering [Ca2+]i in parathyroid cells. The addition of ATP or adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (10-200 microM) to parathyroid cells evoked a rapid and transient increase that was followed by a small, steady-state increase in [Ca2+]i. Cytosolic Ca2+ transients elicited by ATP or ATP gamma S persisted in the absence of extracellular Ca2+ and presence of a mitochondrial uncoupler but were blocked by pretreatment with ionomycin or fluoride. Cytosolic Ca2+ transients elicited by ATP were inhibited by increased concentrations of extracellular Ca2+, Mg2+, or Sr2+. Conversely, ATP depressed increases in [Ca2+]i elicited by these extracellular divalent cations. Parathyroid hormone (PTH) secretion was inhibited by ATP gamma S but not by those nucleotides that were without effect on [Ca2+]i. Loading cells with 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and fura-2 blocked cytosolic Ca2+ transients elicited by ATP gamma S but did not block the inhibitory effects of ATP gamma S on PTH secretion. The results show that the activation of a calcium-mobilizing receptor, in this case by ATP gamma S, is sufficient to inhibit PTH secretion. This favors the view that extracellular Ca2+ acts via a Ca2+-mobilizing receptor to regulate PTH secretion.