1. The effects of diazoxide on ATP-K+ channel currents, recorded from the insulin-secreting cell line, CRI-G1, were studied using patch-clamp techniques. 2. Under current-clamp recording conditions diazoxide (0.6 mM), inhibited action potential activity and hyperpolarized CRI-G1 cells with a concomitant increase in membrane conductance. Recordings from voltage-clamped whole-cells and isolated patches indicate that activation of ATP-K+ channel currents underlie these effects. 3. Diazoxide elicited an activation of ATP-K+ channels which had been partially inhibited by ATP, on application to either surface of the plasma membrane, although it was more effective when applied directly to the cytoplasmic side. Activation of the ATP-K+ currents involves an increase in the single channel open-state probability and an apparent increase in the number of functional channels. 4. Activation was observed only when Mg-ATP was present in the cytoplasmic bathing solution. There was no activation of currents by diazoxide when ATP, in the absence of Mg2+ ions, or Mg-AMP-PNP was present to inhibit the ATP-K+ channels. 5. In the absence of ATP and Mg2+ ions in the cytoplasmic bathing solution, diazoxide (0.6 mM) produced an inhibition of ATP-K+ currents. 6. Cromakalim (BRL 34915) at 10 microM and 100 microM had no significant effects on ATP-K+ currents. 7. It is concluded that diazoxide-induced activation of ATP-K+ channel currents probably involves phosphorylation of the channel or some closely associated membrane protein.