Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.