SUMMARY-enkephalin. This ␦-opioid agonist effect was not observed at FCS concentrations Ͼ0.1% and could be blocked by pretreating cells with pertussis toxin, indicating that G i /G o were involved in this action. In addition, ␦-opioid agonists could potentiate CHO cell proliferation stimulated by those growth factors that are mediated by tyrosine kinase receptors (i.e., insulin, insulin-like growth factor 1, and fibroblast-derived growth factor b). This ␦-opioid agonist potentiation of growth apparently was dependent on the level of ␦-opioid receptors that were expressed and had cell-line selectivity. Activation of ␦-opioid receptors expressed in Rat-1 or NIH3T3 fibroblast did not result in a modulation of the cell growth induced by FCS or by growth factors. Interestingly, in CHO cells transfected with -opioid receptor cDNA, activation with agonists did not produce a potentiation of FCS-stimulated proliferation. This lack of -opioid receptor effect was not due to the differences among CHO clones. In a CHO cell line transfected with both ␦-opioid receptor cDNA and -opioid receptor cDNA, activation of ␦-but not -opioid receptors resulted in a potentiation of growth. These data suggest that ␦-and -opioid receptors in CHO cells activate similar but divergent second messenger pathways, resulting in the differential regulation of cell growth.Numerous studies have indicated that endogenous opioid peptides, products of proenkephalin or pro-opiomelanocortin, inhibit DNA synthesis in in vitro neuronal or glial cultures or in neuroblastoma cells (1-4). These studies were supported by the observation that opioid antagonists could stimulate DNA synthesis and proliferation in some neuroblastoma cell lines. The opioid antagonist, naltrexone, stereoselectively stimulates cell proliferation in murine neuroblastoma NS20Y and N1E-115 cells, human neuroblastoma SK-N-MC cells, and human fibrosarcoma cells (5). This antagonistic effect on NS20Y cells was demonstrated further by the ability of in vivo administration of naltrexone to stimulate the growth of these cells when they were implanted in rodents (6).The suppression effects exhibited by opioid peptide agonists and the stimulatory effects exhibited by naltrexone on neuroblastoma proliferation were rather surprising. By contrast, a substantial volume of evidence suggests that activation of GPCRs could lead to the enhancement of cell proliferation, mitogenesis, and tumorigenicity. Activation of bombesin receptors in Swiss 3T3 fibroblasts and small-cell lung carcinomas resulted in a stimulation of the mitogenic response (7,8). Activation of transfected serotonin receptors (5-HT 1B , 5-HT 1C , 5-HT 2 ), muscarinic receptors, or ␣ 2 -adrenergic receptors in Chinese hamster lung fibroblasts, Rat-1 fibroblasts, or NIH3T3 fibroblasts resulted in increases in DNA synthesis, which are incidents of foci formation and growth rates (9 -13). These positive effects on growth exhibited by GPCRs were demonstrated further by the ability of constitutively active receptor mutants (14)