Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP₃) receptor levels, reduced mitogen-evoked IP₃ formation and Ca²⁺ release, and Ca²⁺ store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca²⁺ release triggered by either ionomycin or by several IP₃-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca²⁺ transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP₃ formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca²⁺ release response to intracellularly applied IP₃. Finally, to compare IP₃ receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP₃ receptor antiserum, Ab⁴⁰. A ~260-kDa band corresponding to the IP₃ receptor protein was noted; its intensity was reduced by ~50% in senescent cells. Thus, we suggest that reduced IP₃ receptor expression, lowered IP₃ formation, and Ca²⁺ release, as well as Ca²⁺ store depletion, all contribute to the deficient Ca²⁺ signaling seen in HDFs undergoing replicative senescence.