The single-channel current recording technique has been used to study the effects of diazoxide, tolbutamide and ATP, separately and combined, on the gating of nucleotide-regulated K+ channels in the insulin-secreting cell line RINm5F. The effects of diazoxide, tolbutamide and ATP4- were studied at the intracellular membrane surface, using the open-cell membrane patch configuration. Alone diazoxide was found only inconsistently to evoke channel stimulation, 57% of all applications of the drug (72 times in 48 separate patches) having no effect at concentrations between 0.02 and 0.4 mM. In the presence of ATP, however, diazoxide consistently evoked channel activation (seen 87 times in 49 patches, 95% of all applications). The interactions of diazoxide and ATP seemed competitive. Stimulation of channels by diazoxide in the presence of 1 mM ATP was suppressed if the concentration of ATP was elevated to 2 or 5 mM. In solutions in which Mg2+ had been chelated with EDTA, diazoxide failed to activate channels closed by 1 mM ATP; however, this was not due to a direct effect on the channels caused by the absence of Mg2+, but could be explained by the enhanced ATP4- concentration after Mg2+ removal. When the total ATP concentration was lowered to give the same [ATP4-] in the absence of Mg2+ to that present in the control experiments, diazoxide was able to evoke full activation. Channel inhibition evoked by tolbutamide, 0.01 to 1.0 mM, did not require the presence of either ATP or Mg2+. In the presence of ATP tolbutamide further reduced the number of channel openings. Diazoxide was able to compete with tolbutamide for control of channel activity, an effect that was augmented by the presence of ATP. In the presence of 0.1 mM tolbutamide, diazoxide was unable to stimulate channel openings; however, if the dose of tolbutamide was lowered or ATP made available to the inside of the membrane, channel stimulation occurred.