Signal-response coupling was studied in an exocrine female accessory sex gland (albumen gland) of the freshwater snail Lymnaea stagnalis. Glands were incubated in vitro with Calfluxin (CaFl), a neuropeptide which stimulates the influx of Ca2+ into the mitochondria of the secretory cells. This influx, which is considered to reflect an increase of Ca2+ in the cytosol, was measured as the percentage mitochondria containing Ca deposits. Ca deposits. Ca deposits were visualized at the ultrastructural level with the pyroantimonate precipitation technique. The origin of the Ca2+ and the mechanism by which the Ca2+ concentration in the cytosol is elevated were investigated. The results indicate that CaFl stimulates the influx of extracellular Ca2+ and mobilizes intracellular Ca2+. The increase of the percentage of mitochondria containing Ca deposits is sensitive to Ca2+ channel blockers (D600, Co2+, La3+), indicating that Ca2+ channels are involved. Li+ ions suppress the CaFl response, which suggests that the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), and thus the production of myo-inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) is involved in the Ca2+ mobilization. The protein kinase-C (PKC) stimulator 4-beta-phorbol 12-beta-myrastate 13-alpha-acetate (PMA) mimicked the response to CaFl. The PKC inhibitors trifluoperazine (TFP) and chlorpromazine (CP) markedly decreased the CaFl-stimulated influx of Ca2+ into the mitochondria. The PMA-stimulated influx of Ca2+ into the mitochondria is not dependent on extracellular Ca2+ and is not sensitive to Ca2+ channel blockers. In PMA-stimulated glands, the Na+/H+ exchange blocker amiloride completely abolished the Ca2+ influx into mitochondria. In CaFl-stimulated glands the influx was partly blocked. Increasing the internal pH of the glandular cells with the Na+/H+ ionophore monensin or with NH4Cl mimicked the CaFl response. It is proposed that upon stimulation with CaFl, mobilization of intracellular Ca2+ is mediated via the PKC-stimulated activation of the Na+/H+ exchange, thus leading to an increase of the internal pH. The role of IP3 in the mobilization of intracellular Ca2+ is uncertain.