The specific binding of a novel tritiated K+ channel opener, [3H]BAY X 9228, has been characterized in a rat insulinoma (RINm5F) cell line. The KD was 2.1 nM and Bmax 50 fmol/mg total protein as determined by saturation analysis. The high affinity binding to intact cells was inhibited by pinacidil and by a series of BAY X 9228 analogs with an activity sequence correlating well with that for producing glyburide-reversible relaxation of partially depolarized rat aorta. This represents the first report of the specific binding of a K+ channel opener to cultured cells.