1. The signal transduction pathway responsible for cAMP-dependent Ca2+-induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores was assessed in the insulin-secreting cell line INS-1. 2. CICR was triggered by the GLP-1 receptor agonist exendin-4, an effect mimicked by caffeine, Sp-cAMPS or forskolin. CICR required influx of Ca2+ through L-type voltage-dependent Ca2+ channels, and was blocked by treatment with nimodipine, thapsigargin, or ryanodine, but not by the IP3 receptor antagonist xestospongin C. 3. Treatment with the cAMP antagonist 8-Br-Rp-cAMPS blocked CICR in response to exendin-4, whereas the PKA inhibitor H-89 was ineffective when tested at a concentration demonstrated to inhibit PKA-dependent gene expression. 4. RT-PCR of INS-1 cells demonstrated expression of mRNA coding for the type-II isoform of cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF-II, Epac2). 5. CICR in response to forskolin was blocked by transient transfection and expression of a dominant negative mutant isoform of cAMP-GEF-II in which inactivating mutations were introduced into the exchange factor's two cAMP-binding domains. 6. It is concluded that CICR in INS-1 cells results from GLP-1 receptor-mediated sensitization of the intracellular Ca2+ release mechanism, a signal transduction pathway independent of PKA, but which requires cAMP-GEF-II.