Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on adrenal medullary (AM) cells as a neurotransmitter of the sympathetic nerve. We have elucidated that PACAP facilitates catecholamine secretion evoked by nAChR or mAChR stimulation and PACAP itself induces a depolarizing inward current in guinea-pig AM cells (Inoue et al., 2000). The ionic mechanism of PACAP-induced currents still remains unclear. Thus, the present experiments are aimed first to investigate the ion channels involved in the PACAP-sensitive current and second to explore whether or not the regulation of ion channels by PACAP is preserved in AM cells of guinea pigs and mice. After the animals were subjected to cervical dislocation, the adrenal gland was taken away and the adrenal cortex was removed from the adrenal medulla. After collagenase treatment, AM cells were mechanically isolated in a bath apparatus. The whole-cell current was recorded at 26-29°C with the perforated patch clamp technique. Application of 3 nM PACAP and 1 mM muscarine produced inward currents in 6 and 8 of 9 guinea-pigs AM cells, respectively, whereas it induced an enhancement of the muscarinic currents in all the cells examined, respectively. The 1 mM muscarine-induced currents were enhanced by 122 ± 44% (n = 9) in the presence of 3 nM PACAP, and the extent of enhancement of the muscarinic current did not depend upon the amplitude of PACAP-induced currents. In contrast to guinea pigs, almost all the mouse AM cells (14/15) developed inward currents in response to 3 nM PACAP, whereas only 30% of the mouse cells exhibited inward currents in response to 30 mM muscarine. In guinea-pig and mouse AM cells, 1 mM mucarine-induced currents, which are due to activation of nonselective cation (NS) channels (Inoue et al., 2012), were suppressed by quinine in a dose-dependent manner with an IC50 of 20 mM, whereas PACAP-induced inward currents were not noticeably suppressed by 100 mM quinine. The PACAP current was completely suppressed by the replacement of external Na+ with N-methyl D-glucamine. Addition of 100 mM Zn2+ to the external solution resulted in the inhibition of PACAP currents by 57%. Similar extents of suppression were noted with a decrease in external pH to 6.4. The present results reveal that PACAP activates Na+-permeable cation channels, which are not affected by quinine. The PACAP-regulated cation channel is suppressed by external Zn2+ or a decrease in external pH. These properties of the ion channels are reminiscent of those of TRPM4 or TRPM5. We conclude that PACAP activates Na+-permeable cation channels in guinea-pig and mouse AM cells, which differ from NS channels coupled with mAChRs.