SK4/IK1 encodes an intermediate conductance, Ca²⁺-activated K⁺ (KCa) channel and fulfills a variety of physiological functions in excitable and non-excitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channel in salivary acinar cells, either regulatory mechanism or physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, K_Ca currents in macropatches excised from RSA cells matched those of whole-cell currents recorded from HEK293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg²⁺ were present in the pipette solution. The nonhydrolysable ATP analogue, AMP-PNP failed to support the channel activity as ATP did. Addition of Rp-cAMPS (10 µM), a PKA inhibitor, to the pipette solution containing ATP/Mg²⁺ induced run-down of the KCa currents. Inclusion of cAMP (1 mM) into the pipette solution (1 µM free Ca²⁺) containing ATP/Mg²⁺ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 µM free Ca²⁺. Forskolin (1 µM), an adenylyl cyclase activator, also increased the currents induced by 0.1 µM free Ca²⁺. In inside-out macropatches, cytosolic ATP/Mg²⁺ increased both the maximum current (i.e. the maximum channel activity) and Ca²⁺-sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.