We previously reported (Robbins et al., 2006) that expression of Kir2.1 potassium channels in sympathetic neurons reduced the amplitude of endogenous potassium M-currents (IM, carried by Kv7.2/7.3 channels). Since both channels require membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to open, and since the apparent ‘affinity’ of Kir2.1 channels for PIP2 is ~20 times greater than that of Kv7.2/7.3 channels (Lopes et al., 2002; Li et al., 2005), we suggested that Kir2.1 might reduce IM by out-competing it for available PIP2. We have now examined this further, using neurons isolated from rat superior cervical ganglia in short-term tissue culture. Membrane currents were recorded using perforated-patch electrodes. A voltage ramp-and-hold protocol (Robbins et al, 2006) was used to simultaneously measure Kir2.1 and KM currents. IKir was measured at -130 mV, IM as amplitude of deactivation tail-currents on stepping from -20 to -50 mV after subtracting residual current in 10-30 µM linopirdine. cDNAs were expressed by intranuclear injection with green fluorescent protein (GFP) cDNA marker and cells tested 24 hr later. Principal results were as follows: (1). In confirmation of previous observations, 24 hr expression of Kir2.1 cDNA generated an inward-rectifier current of 144.7±22 pA.pF-1 at -130 mV and reduced IM from 5.91 ± 0.33 to 2.86 ± 0.43 pA.pF-1(mean ± SEM, n = 7 for each; P<0.01, Dunnett’s test). (2). Reduction of IM was accompanied by a 5-fold increase in its sensitivity to inhibition by a PIP2-sequestering peptide (see Robbins et al., 2006) (IC50 mean ± SEM: control 1.63 ± 0.22 µM; + peptide 0.31 ± 0.07, each n = 6; P<0.01, Tukey’s test). (3). Expression of some mutated Kir2.1 channels that show less apparent affinity for PIP2 produced less effect on IM. Thus, Kir2.1[R228Q] (Lopes et al., 2002) produced no significant reduction of IM or change in PIP2-sequestering peptide sensitivity. (4). Membrane targeting of an expressed PIP-kinase that increases membrane PIP2 (Suh et al., 2006) increased IM in Kir2.1-expressing neurons (+12.6 ± 2.8 %, n=10) but not in control neurons (+1.04 ± 1.0 %, n=5; P for difference < 0.01, Tukey’s test), hence partly reversing the effect of Kir2.1. Expression of a kinase-inactive construct had no effect on IM under either condition. (5). Kir2.1 expression had no significant effect on the overall cell level of Kv7.2 protein assessed by single-cell Kv7.2 antibody immunohistochemistry. These results support the ‘PIP2 competition’ hypothesis. They may also partly explain why sympathetic neurons that exhibit large M-currents do not have constitutive inward-rectifier currents, and vice-versa (Wang & McKinnon, 1995).