In the present study we determined the mRNA-expression of pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptors in primary cultures of rat cortical neurons and type I astrocytes, and investigated the effects of PACAP38 on adenylyl cyclase, inositol phospholipid hydrolysis and intracellular calcium homeostasis. PACAP38 elicited a concentration-dependent (1 n m–100 nm) increase in inositol phosphate levels and [Ca2+]i in neurons but not in type I astrocytes. The PACAP-induced increase of intracellular calcium concentration, [Ca2+]i, was characterized by a spike, compatible with inositol trisphosphate (IP3) -induced calcium mobilization from intracellular stores, and a plateau phase, sustained by activation of capacitative calcium entry triggered by depletion of IP3-sensitive calcium stores. In the absence of extracellular calcium, only the spike phase was present while the plateau phase was clearly reduced. In addition, thapsigargin pretreatment abolished the PACAP38-induced [Ca2+]i rise. Treatment with 1 μm VIP did not affect [Ca2+]i in either neurons or type I astrocytes, clearly indicating the coupling of PAC1–HOP subtype to phospholipase-C in neurons. In addition, as previously reported, PACAP38 stimulated cAMP formation in both neurons and type I astrocytes. Using the reverse transcription polymerase chain reaction, we found mRNA-expression of PAC1 (PACAP – HOP variant) and VPAC2 in neurons, PAC1 (PACAP – R variant), VPAC1 and VPAC2 in astrocytes. These data indicate both a functional and molecular diversity of PACAP and VIP receptors in these cell types and support the view that the PAC1-HOP variant may be responsible for phospholipase-C activation and [Ca2+]i elevation in cortical neurons.