The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11κ7 cell line (an immortalized mouse neuroblastoma × dorsal root ganglion hybrid stably transfected to overexpress κ-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the μ-specific opioid agonist morphiceptin (E7CH) or the κ-specific opioid agonist U69,593 (F-11κ7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with non-pretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16-24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp-adenosine 3′,5′-cyclic monophosphothioate and increased killing with the antagonist Rp-adenosine 3′,5′-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.