Previous evidence has suggested a role for calmodulin (CaM) in opioid receptor signaling. We demonstrate here that morphine stimulation of the mu-opioid (OP3) receptor causes rapid CaM translocation to the nucleus in OP3-transfected human embryonic kidney (HEK)-293 cells and in SH-SY5Y human neuroblastoma cells. Ca2+ influx into the cells resulting from OP3 receptor activation was required for nuclear CaM translocation. Moreover, in HEK-OP3 and SH-SY5Y cells, increased nuclear CaM content was associated with enhanced phosphorylation of the nuclear transcription factor cyclic AMP-responsive element-binding protein. This appeared to be mediated by Ca2+/CaM kinases and also by a pathway involving protein kinase C. CaM was previously shown to bind directly to the OP3 receptor and to be released from the plasma membrane on agonist stimulation. To test whether OP3-mediated CaM release contributes to nuclear CaM signaling, we used a mutant OP3 receptor (K273A) with reduced affinity for CaM that fails to release CaM from the plasma membrane. K273A-OP3 activated Ca2+ influx to a similar extent as wild-type OP3; however, CaM translocation to the nucleus was attenuated. These results indicate that OP3-stimulated Ca2+ influx results in nuclear CaM translocation, which appears to be enhanced by simultaneous CaM release by OP3 wild-type receptor from plasma membranes. These results suggest a novel Ca2+/CaM signaling pathway of opioid receptors in the regulation of transcriptional activity.