We have identified the single PAC1 receptor variant responsible for Ca²⁺ mobilization from intracellular stores (ICS) and influx through voltage-gated Ca²⁺ channels (VGCCs) in bovine chromaffin cells (BCCs), and the domain of this receptor variant that confers coupling to [Ca²⁺]_i elevation. This receptor (bPAC1_hop) contains a 28 amino acid ‘hop’ insertion in the third intracellular loop, with a full length 171 amino acid N-terminus. Expression of the bPAC1_hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding, PACAP-dependent elevation of both cAMP and intracellular Ca²⁺ concentrations [Ca²⁺]_i. Removal of the hop domain and expression of this receptor (bPAC1_null) in NG108-15 cells reconstituted high-affinity PACAP binding and PACAP-dependent cAMP generation, but without a corresponding [Ca²⁺]_i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation, but do not express PAC1 receptors at levels found in post-mitotic neuronal and endocrine cells, and do not support PACAP-mediated neurosecretion. Expression of bPAC1_hop, but not bPAC1_null, at levels comparable to those of bPAC1_hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca²⁺]_i increase, and extracellular Ca²⁺ influx. In addition, PC12-G cells expressing bPAC1_hop acquired the ability to release [³H]-norepinephrine in a Ca²⁺–influx dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than non-neuroendocrine cells, therefore reveals key differences between PAC1_hop and PAC1_null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca²⁺-signaling in neuroendocrine cells.