Tamoxifen inhibits transmembrane currents of the Kir2.x inward rectifier potassium channels by interfering with the interaction of the channels with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂). We tested the hypothesis that Kir channels with low affinity for PIP₂, like the adenosine triphosphate (ATP)-sensitive K⁺ channel (K_ATP) and acetylcholine (ACh)-activated K⁺ channel (K_ACh), have at least the same sensitivity to tamoxifen as Kir2.3. We investigated the effects of tamoxifen (0.1 – 10 μM) on Kir6.2/SUR2A (K_ATP) and Kir3.1/3.4 (K_ACh) channels expressed in HEK-293 cells and ATP-sensitive K⁺ current (I_KATP) and ACh-activated K⁺ current (I_KACh) in feline atrial myocytes. The onset of tamoxifen inhibition of both I_KATP and I_KACh was slow (T_1/2 approximately 3.5 min) and concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. Tamoxifen interacts with the pore forming subunit, Kir6.2, rather than with the SUR subunit. The inhibitory potency of tamoxifen on the Kir6.2/SUR2A channel was decreased by the mutation (C166S) on Kir6.2 and in the continuous presence of PIP₂. In atrial myocytes, the mechanism and potency of the effects of tamoxifen on K_ATP and K_ACh channels were comparable to those in HEK-293 cells. These data suggest that, similar to its effects on Kir2.x currents, tamoxifen inhibits K_ATP and K_ACh currents by interfering with the interaction between the channel and PIP₂.