Stimulation of changes in cytosolic free calcium by parathyroid hormone was determined in three opossum kidney (OK) cell types, OK wild-type, OKP clone, and OKH clone. All three types of OK cells express parathyroid hormone (PTH)-sensitive adenylate cyclase and adenosine 3',5'-cyclic monophosphate (cAMP) production. However, only the OK wild-type and the OKP clone respond to PTH with inhibition of sodium-dependent Pi transport and transient increase in cytosolic calcium. Characterization of the increases in cytosolic calcium in the wild-type and OKP clones revealed they were due in part to stimulation of Ca2+ release from intracellular stores, probably by inositol 1,4,5-trisphosphate (IP3), which was stimulated by PTH. PTH-stimulated Ca2+ transients were also inhibited by protein kinase C activation. These data are compatible with PTH receptor-mediated phospholipase C activation and its feedback inhibition by protein kinase C. The OKH cells demonstrated a slow increase in cytosolic calcium when stimulated by cyclic nucleotides but no evidence for PTH stimulation of Ca2+ release from intracellular stores. Thus the absence of an inhibitory response of sodium-dependent Pi transport to PTH in the OKH cells is associated with the absence of the rapid transient elevations of cytosolic Ca2+ such as those produced by IP3 production. These data suggest an important cooperative role for cAMP and the phospholipase C-stimulated Ca2(+)-protein kinase C message system in the regulation of Pi transport.