In order to study signal transduction in PACAP-stimulated GH release, effects of ovine PACAPs (oPACAPs) on cAMP synthesis and intracellular free Ca(2+)([Ca(2+)](i)) levels in primarily cultured common carp (Cyprinus carpio) pituitary cells were investigated using radioimmunoassay (RIA) for cAMP and [Ca(2+)](i) Indo-1/AM loading techniques. The main results obtained could be briefly summarized as follows: parallel with their GH-releasing effects, oPACAP-38 (0.01 nmol/L to 1 micromol/L) and oPACAP-27 (0.01 nmol/L to 1 micromol/L) were also effective in increasing cAMP release, cellular cAMP content, and total cAMP production in a dose-dependent manner in common carp pituitary cells. In addition to their cAMP synthesis, 0.5 micromol/L oPACAP-38 and 0.5 micromol/L oPACAP-27 were also effective in elevating [Ca(2+)](i) levels in common carp pituitary cells preloaded with the Ca(2+)-sensitive dye Indo-1; the stimulatory actions were dependent on the presence of extracellular Ca(2+) ([Ca(2+)](e)), as removal of [Ca(2+)](e) by adding th e Ca(2+) chelator 4 mmol/L E GTA blocked the rise of [Ca(2+)](i) induced by oPACAP-38 and oPACAP-27, respectively. Besides, the rise in [Ca(2+)](i) induced by 0.5 micromol/L oPACAP-38 was b locked by treatment with 10 micromol/L nifedipine, an inhibitor of "L-type" voltage-sensitive Ca(2+) channels (VSCC); but 0.5 micromol/L oPACAP-38 was not effective in elevating [Ca(2+)](i) levels after the cells treated with Ca(2+)-sensitive dye Indo-1 were challenged with 10 micromol/L nifedipine. These results, taken as a whole, strongly suggest the involvement of the cAMP- and Ca(2+)-dependent pathways in PACAP-stimulated GH release in common carp.