||In mammals, the G protein-coupled opioid receptors are mainly expressed in the central nervous system, and early pharmacological studies on immunocytes indicate that the classical δ-, μ- and κ-opioid receptors are also present in platelets, lymphocytes, macrophages and monocytes. The biological effects exhibited by opioid receptors on the nervous and immune systems are diverse and complicated; they are involved in both stimulatory and inhibitory regulations on proliferation, differentiation and even apoptosis. Some of these physiological processes are also mediated by mitogen-activated protein kinases (MAPKs). Many studies have shown that activated opioid receptors may contribute to enhanced MAPK activity, so the opioid receptors-regulated the activity of c-Jun NH2-terminal kinases (JNKs) is also plausible, and this regulation may play in functional responses in neuronal cells and immunocytes. By using COS-7 cells transiently transfected with the cDNAs of the opioid receptors and HA-tagged JNK, the signaling components involved in this pathway was delineated. It was demonstrated that the three opioid receptors could significantly stimulate JNK activity in a PTX-sensitive and Gβγ-dependent manner. Moreover, the receptors regulated JNK activity through Src family tyrosine kinases, Rac and Cdc42 GTPases, but they do not require EGF receptors, Ras and Rho. Interestingly, a distinct difference between the μ- and δ- or κ-opioid receptors signalings were observed, whereby PI3K was only necessary for the signaling of μ- opioid receptors, but not δ- and κ-opioid receptors. The present study also provided evidences that opioid receptors-induced JNK activation similarly occur in human neuroblastoma SH-SY5Y cells, NG 108-15 neuroblastoma x glioma hybrid cells and human monocytic THP-1 cells that endogenously express μ-, δ- and κ-opioid receptors, respectively. The major signaling properties of JNK stimulation by the opioid receptors were thus further confirmed under more physiological conditions.