Nitric oxide (NO) is assumed to contribute to the impairment of B-cell function in type 1 diabetes mellitus (IDDM). In the present paper we show that in mouse B-cells with intact metabolism authentic NO (20 μM) led to a biphasic effect on the K⁺_ATP current, namely a transient increase and a consecutive almost complete inhibition. This resembles closely the effect that we have observed previously with the NO donor S-nitrosocysteine (SNOC, 1 mM) suggesting that merely NO caused both phases of this effect. We now demonstrate that the rise in the current amplitude was accompanied by a depolarization of the mitochondrial membrane potential ΔΨ and a concomitant reduction in the ATP/ADP ratio. Thus, it seems likely that the increase in current amplitude is due to the interference of NO with cell metabolism. The subsequent inhibition of the K⁺_ATP current is assumed to be caused by a direct effect on the channel since K⁺_ATP single channel current activity measured in excised patches was strongly reduced by authentic NO and SNOC. Our data reveal new insights into the mechanisms underlying the biphasic action of NO on K⁺_ATP channels in pancreatic B-cells.