Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar signaling pathway is used by the mu-opioid receptor (MOR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation of MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosphorylation. Data obtained with inhibitors of EGFR Tyr kinase and membrane metalloproteases support an intermediate role of EGFR activation, involving release of endogenous membrane-bound epidermal growth factor. Previous studies had demonstrated a role for CaM in opioid signaling based on direct CaM binding to MOR. To test whether CaM contributes to EGFR transactivation and ERK phosphorylation by MOR, we compared wild-type MOR with mutant K273A MOR, which binds CaM poorly, but couples normally to G proteins. Stimulation of K273A MOR with [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (10-100 nm) resulted in significantly reduced ERK phosphorylation. Furthermore, wild-type MOR stimulated EGFR Tyr phosphorylation 3-fold more than K273A MOR, indicating that direct CaM-MOR interaction plays a key role in the transactivation process. Inhibitors of CaM and protein kinase C also attenuated [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin-induced EGFR transactivation in wild-type (but not mutant) MOR-expressing cells. This novel pathway of EGFR transactivation may be shared by other G protein-coupled receptors shown to interact with CaM.