Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)‐induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5‐bisphosphate (PIP2). SP rapidly – with a half‐time of ∼10 s with intracellular GTPγS and ∼14 s with intracellular GTP – inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP2 than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP2. A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP2 than wild type Kir3.1/Kir3.2, shows a SP‐induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP2 theory of channel inhibition. Co‐immunoprecipitation reveals that Gαq binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co‐immunoprecipitation data suggest that Gq activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Gαq to the channel.