Parathyroid hormone (PTH) produced a dose-dependent immediate stimulation of inositol triphosphate and diacylglycerol production in the opossum kidney cell line, primary culture proximal tubular cells, and basolateral membranes from canine proximal tubular segments. The increase in inositol triphosphate production was accompanied by a minor increase in inositol phosphate and no significant increase in inositol bisphosphate production. Associated with the changes in inositol triphosphate and diacylglycerol, there was an immediate hydrolysis of phosphatidylinositol 4,5-bisphosphate. The effect on phospholipid hydrolysis was followed by stimulation of phosphorylation of phosphatidylinositol 4' monophosphate and phosphatidylinositol. PTH produced a sudden increase in cytoplasmic Ca2+ in opossum kidney cells that persisted for ~1 min. Inositol triphosphate transiently increased cytoplasmic Ca2+ in saponin-treated opossum kidney and primary culture proximal tubule cells. The effects of PTH were not mimicked by cyclic nucleotides. In fact, cyclic AMP appeared to diminish inositol triphosphate production. These results demonstrate that PTH may activate renal tubular epithelial cells by the production of inositol triphosphate and diacylglycerol.