Activation by opioid receptors of cell proliferation was examined with fibroblast cell lines stably expressing either δ-opioid or μ-opioid receptors. Addition of [D-Ala2,D-Leu5]-enkephalin or [d-Pen2,D-Pen5]- enkephalin to Chinese hamster ovary (CHO) cells transfected with δ-opioid receptor cDNA resulted in an agonist concentration-dependent potentiation of fetal calf serum (FCS)-stimulated cell proliferation. This potentiation by δ-opioid agonists was antagonized by naloxone and was not observed with the κ-opioid receptor selective agonist U50,488 or the μ-opioid receptor selective agonist [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin. This δ-opioid agonist effect was not observed at FCS concentrations >0.1% and could be blocked by pretreating cells with pertussis toxin, indicating that G(i)/G(o) were involved in this action. In addition, δ-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 δ-opioid agonist potentiation of growth apparently was dependent on the level of δ-opioid receptors that were expressed and had cell-line selectivity. Activation of δ-opioid receptors expressed in Rat-1 or NIH3T3 fibroblast did not result in a modulation of the celt growth induced by FCS or by growth factors. Interestingly, in CHO cells transfected with μ-opioid receptor cDNA, activation with agonists did not produce a potentiation of FCS-stimulated proliferation. This rack of μ-opioid receptor effect was not due to the differences among CHO clones. In a CHO cell line transfected with both δ- opioid receptor cDNA and μ-opioid receptor cDNA, activation of δ-but not μ-opioid receptors resulted in a potentiation of growth. These data suggest that δ- and μ-opioid receptors in CHO cells activate similar but divergent second messenger pathways, resulting in the differential regulation of cell growth.