δ-Opioid receptor (DOR)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is mediated by the transactivation of epidermal growth factor (EGF) receptors. Here we demonstrate that in stably DOR-expressing human embryonic kidney (HEK) 293 (HEK/DOR) cells, down-regulation of EGF receptors by long-term EGF (0.1 μg for 18 h) treatment, but not by small interfering RNA, results in functional desensitization of EGF (10 ng/ml)-stimulated ERK1/2 signaling. In EGF receptor-desensitized (HEK/DOR(-EGFR)) cells, however, [d-Ala²,d-Leu⁵]enkephalin (1 μM) and etorphine (0.1 μM) retained their ability to stimulate ERK1/2 activation. The newly acquired signal transduction mechanism is insensitive to the EGF receptor blockers 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), does not involve DOR internalization and activation of the focal adhesion kinase pp125FAK, but requires matrix metalloproteinase-dependent release of soluble growth factors. A supernatant transfer assay in which conditioned growth media of opioid-treated HEK/DOR and HEK/DOR(-EGFR) "donor" cells are used to stimulate ERK1/2 activity in DOR-lacking HEK293 wild type and HEK293(-EGFR) "acceptor" cells revealed that long-term EGF treatment produces a switch in the receptor tyrosine kinase (RTK) system transactivated by opioids. Using microfluidic electrophoresis, chemical inhibitors, phosphorylation-specific antibodies, and EGF receptor-deficient Chinese hamster ovary-K1 cells, we identified the release of an insulin-like growth factor-1 (IGF-1)-like peptide and activation of IGF-1 receptors in HEK/DOR(-EGFR) cells after DOR activation. A similar switch from a neurotrophic tyrosine kinase receptor type 1 to an IGF-1 receptor-dependent ERK1/2 signaling was observed for chronically nerve growth factor-treated neuroblastoma × glioma (NG108-15) cells. These results indicate that transactivation of the dominant RTK system in a given cellular setting may represent a general feature of opioids to maintain mitogenic signaling.