Although the Ca2+-activated K+ (IK,Ca) channel is known to play an important role in the maintenance of resting membrane potential, the regulation of the channel in physiological condition is not completely understood in vascular myocytes. In this study, we investigated the role of cytoplasmic Mg2+ on the regulation of IK,Ca channel in pulmonary arterial myocytes of the rabbit using the inside-out patch clamp technique. Mg2+ increased open probability (Po), but decreased the magnitude of single channel current. Mg2+-induced block of unitary current showed strong voltage dependence but increase of Po by Mg2+ was not dependent on the membrane potential. The apparent effect of Mg2+ might, thus, depend on the proportion between opposite effects on the Po and on the conductance of IK,Ca channel. In low concentration of cytoplasmic Ca2+, Mg2+ increased IK,Ca by mainly enhancement of Po. However, at very high concentration of cytoplasmic Ca2+, such as pCa 5.5, Mg2+ decreased IK,Ca through the inhibition of unitary current. Moreover, Mg2+ could activate the channel even in the absence of Ca2+. Mg2+ might, therefore, partly contribute to the opening of IK,Ca channel in resting membrane potential. This phenomenon might explain why IK,Ca contributes to the resting membrane potential where membrane potential and concentration of free Ca2+ are very low.