1. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were examined using the patch clamp technique in the whole-cell, cell-attached and inside-out patch configurations. 2. The cells transfected with IRK2 cDNA exhibited a K+ current which showed classical properties of inwardly rectifying K+ channels at both whole-cell and single-channel levels. 3. In the inside-out patch configuration, intracellular Mg2+ (Mg2+i blocked the outward currents in a voltage-dependent and virtually time-independent manner. Mg2+i (1-100 microM) caused a decrease in the unitary current amplitude of the IRK2 channel by inducing subconducting levels. 4. In the absence of Mg2+i, intracellular spermine blocked the outwardly flowing IRK2 currents in a voltage- and time-dependent manner. Spermine (1-100 nM) did not affect the unitary channel current amplitude but reduced the channel open probability. The spermine block showed a slower time and steeper voltage dependence than the Mg2+i++ block. 5. When both these blockers were present, Mg2+i apparently attenuated the inhibitory effect of spermine on the outwardly flowing IRK2 currents. This interaction was voltage and time dependent, and could be well explained by a model in which Mg2+i and spermine competitively bind to the channel with their individual first-order kinetics. This competition would induce time-dependent transits of the channel between the Mg2+i -and spermine-blocked states via a single open state, thereby preserving a certain size of persistent outward currents at depolarized potentials.