We investigated the block of KATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited KATP channels with an apparent Ki of 63 nm and a Hill coefficient of 0.85. The inhibition of KATP channels by glibenclamide was unaffected by internal Mg2+. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased.(3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and KD to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 x 10(7) m-1 sec-1 and an unbinding rate of 6.26 sec-1. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 x 10(6) m-1 sec-1.