1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 ± 2.0 or 3.5 ± 0.5 μg ml-1 (corresponding to 3.7 ± 0.9 or 1.7 ± 0.2 μmol l-1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3. Dose dependency studies of GIRK activation by purified βγ subunits of the G protein (G(βγ)) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G(βγ). 4. Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5. Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6. These data support the hypothesis that the distal part of the carboxy-terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of G(βγ).