While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.