The inwardly rectifying potassium channel Kir2.3 possesses extracellular cysteine residues at positions 113, 140, and 145, as well as at position 79 near the outer membrane boundary. In this study, we have investigated the roles of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenesulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine residues were mutated to serine by site-directed mutagenesis. Wild-type or mutant cRNA was injected into Xenopus oocytes and voltage-clamp recordings made 1-2 days later. Wild-type Kir2.3 currents were significantly inhibited by PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S were also inhibited by PCMBS, thimerosal and H2O2. These mutations affected the time course of inhibition by all three reagents. For PCMBS, a slow component of inhibition was absent for the C79S mutation, and a fast component was absent for C140S. For the double mutation C79S/C140S, PCMBS no longer had any effect. For thimerosal, there was a slower time course for C140S, a faster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, the main effect was a delayed onset for the double mutant. The reducing agent dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2. Finally, wild-type Kir1.1 currents were not significantly inhibited by the applications of either PCMBS or thimerosal, while H2O2 produced small inhibition. The results taken together indicate that inhibition by the cysteine-reactive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2.3 channels, with C79 mediating a slow component of inhibition and C140 a faster component, and that both residues are extracellularly exposed. The data indicate that these two cysteine residues are also main sites for inhibition by thimerosal and H2O2 but, unlike for PCMBS, an additional non-extracellular inhibitory site(s) must also be involved.