Abstract We studied the block of whole-cell ATP-sensitive K+ (KATP) currents in mouse pancreatic -cells produced by external Ba2+. Ba2+ produced a time- and voltage-dependent block of KATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6mM [K+]o, the relationship between the steady-state KATP current and [Ba2+]o was fit by the Hill equation with a Kd of 12.52.8M at 123mV and of 0.180.02mM at 62mV. The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba2+ ion is sufficient to block the channel. When [K+]o was raised to 28mM the Kd was little changed (12.44.1M at 123mV, 0.270.05mM at 62mV) and n was unaffected, suggesting that K+ does not interact with the Ba2+ binding site. The kinetics of Ba2+ block were slow, 10M Ba2+ blocking the KATP current with a time constant of 20ms at 123mV in 28mM [K+]o. The blocking rate constant was calculated as 1.7mM1ms1 and the unblocking rate as 0.02ms1, at 123mV. The data are discussed in terms of a model in which Ba2+ binds to a site at the external mouth of the channel to inhibit the KATP channel.