Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca(2+) channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca(2+) release, and the resulting depletion of the ER store, triggers entry of extracellular Ca(2+) by store-operated Ca(2+) entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, we examined the effects of mutations in the IP3R gene (itpr) on Ca(2+) signals in cultured neurons derived from Drosophila pupae. We observed increased spontaneous Ca(2+) influx across the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca(2+) influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca(2+) channel and the ER Ca(2+)-sensor that regulates it, respectively. Expression of voltage-gated Ca(2+) channels (cac, Ca-α1D and Ca-αT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca(2+) influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca(2+) influx and expression of PM Ca(2+) channels in Drosophila pupal neurons. Spontaneous Ca(2+) influx compensates for the loss of SOCE in Drosophilaitpr mutant neurons.