This study determined the regulatory effect of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on the basal Ca2+ transients in cardiomyocytes. In cultured neonatal rat ventricular myocytes (NRVMs) at different densities, we used confocal microscopy to assess the effect of IP3Rs on the endogenous spontaneous Ca2+ oscillations through specific activation of IP(3)Rs with myo-IP3 hexakis (butyryloxymethyl) ester (IP3BM), a membrane permeable IP3, and interference of IP3R expression with shRNA. We found that NRVMs at the monolayer state displayed coordinated Ca2+ transients with less rate, shorter duration, and higher amplitude compared to single NRVMs. In addition, monolayer NRVMs exhibited 4 or 10 times more increased Ca2+ transients in response to phenylephrine, an a-adrenergic receptor agonist, or IP3BM than single NRVMs did, while the transient pattern remained unaltered, suggesting that the sensitivity of intracellular Ca2+ response to IP3R activation is different between single and monolayer NRVMs. However, interference of IP3R expression with shRNA reduced the frequency and amplitude of the spontaneous Ca2+ fluctuates similarly in both densities of NRVMs, resembling the effects of ryanodine receptor inhibition by ryanodine or tetracaine. Our findings suggest that IP(3)Rs are involved, in part, in the regulation of native Ca2+ transients, in profiles of their initiation and Ca2+ release extent, in developing cardiomyocytes. In addition, caution should be paid in evaluating the behavior of Ca2+ signaling in primary cultured cardiomyocytes at different densities.