To investigate the characteristics of large-conductance calcium-activated potassium channels (BK(Ca)) and the effect of hydrogen peroxide (H2O2) on BK(Ca) in guinea-pig proximal colon smooth muscle cells, single smooth muscle cells of guinea-pig colon were enzymatically isolated in low calcium solution containing papain (3 mg/mL), DTT (2 mg/mL), and bovine serum albumin (BSA, 2 mg/mL). Tissues were incubated at 36 degrees C in enzyme solution for 15 min and were then suspended in enzyme-free low calcium solution. Inside-out single channel recording technique was used to record BK(Ca) current. The intracellular (bath) and microelectrode solution both contained symmetrical high potassium. The BK(Ca) in guinea-pig colon smooth muscle cell possesses: 1) voltage-dependence, 2) high selectivity for potassium ion, 3) large conductance (223.7 pS+/-9.2 pS), 4) dependence of [Ca(2+)](i). Intracellular application of H2O2 decreased the open probability (P(o)) of BK(Ca) at low concentration (</=1 mmol/L), and increased or decreased P(o) of BK(Ca) at high concentration (5 mmol/L), without affecting the unitary conductance. The effects of H2O2 were reversed by reducing agent dithiothreitol (DTT). Similarly, cysteine specific oxidizing agent, DTNB, also increased or decreased P(o) of BK(Ca) and DTT partially reversed the effect of DTNB. It is thus suggested that H2O2 and DTNB may modulate P(o) of BK(Ca) via the oxidation of cysteine residue.