The PACAP-selective PAC1 receptor (Adcyap1r1) is a G protein coupled receptor (GPCR) that activates both adenylyl cyclase and phospholipase C. Similar to many other GPCRs, our previous studies have shown that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a HEK293 PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization as determined by treatment with pharmacological inhibitors of endocytosis or reducing temperature, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell permeable cAMP analogues bromo-cAMP and dibutyryl-cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than by the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms in addition to PAC1 internalization/endosome activation were involved. Inhibition of PAC1 receptor-stimulated PLC/DAG/PKC signaling with BimI also attenuated ERK phosphorylation and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. The inhibition of both PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/DAG/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.