Cs+ block of GIRK1/GIRK4 expressed in Xenopus oocytes has been investigated. It has been reported that a negatively charged aspartate residue at position 172 in IRK1 is responsible for Cs+ block of the channel. IRK1, a homotetramer, has four aspartate residues at this position. GIRK1/GIRK4 is a heterotetramer and has two aspartate residues at the equivalent position (GIRK1-D173) and, consequently, it should be less sensitive to Cs+. Cs+ caused voltage-dependent block of GIRK1/GIRK4 current (measured with the two-microelectrode voltage-clamp technique). The apparent fraction of the electrical field through which Cs+ moves in order to reach its site of block (delta approximately equals 1.66) is comparable to that in IRK1, suggesting that Cs+ binds to a similar site in the two channels. GIRK1/GIRK4 was less sensitive than IRK1 to Cs+ -the Kd was 3.0-8.5 times greater and at potentials more negative than approximately or = to 130 mV there was voltage-dependent relief of block of GIRK1/GIRK4 (not the case with IRK1). However, the mutations GIRK1-D173A and GIRK1-D173Q increased the sensitivity of the channel to Cs+, while adding a negatively charged aspartate residue to GIRK4 at the equivalent position (GIRK4-N 79D) decreased Cs+ sensitivity. GIRK1-D173 cannot be the site of Cs+ block of GIRK1/GIRK4.