Objective: Although the existence of ATP-sensitive K+ (KATP) channels in vascular muscle is widely accepted, there appears to be little consensus as to what the primary regulator of these channels is under physiological or pathophysiological conditions. Recent evidence has suggested that nucleotide diphosphates (NDPs) may play a more important role than ATP. However, since the properties of vascular KATP channels are quite diverse, and the effects of these nucleotides are poorly understood, the aim of this study was to test the hypothesis that intracellular ATP can regulate whole-cell KATP current (IkATP) in the absence of NDPs. Methods: Single cells were isolated from rabbit main pulmonary artery by enzymatic treatment with papain. Whole-cell patch clamp experiments were performed in cells dialysed with different nucleotides. The effect of the KATP channel activator, levcromakalim (10 μM), was investigated at a holding potential of −60 mV. The contribution of IK,ATP to the holding current was defined as the current which was blocked by glibenclamide following washout of levcromakalim. Results: Lowering the intracellular ATP concentration ([ATP]i) from 1 to 0.1 mM, in the presence or absence of GTP, enhanced the levcromakalim-induced current (Ilev) by ~2.5 fold and increased a glibenclamide-sensitive background K+ current (Iglib). However, Iglib was larger with GTP and the total glibenclamide-sensitive current (Ilev + Iglib) increased with time. Significant activation of Iglib failed to occur when the pipette contained no nucleotides and the responses to levcromakalim were generally much smaller than seen with ATP. GDP (0.5 mM), in the absence of pipette ATP, activated a large background K+ current which had similar properties to Ilev. Consistent with this was the observation that Ilev became substantially reduced in the presence of GDP, presumably because a significant amount of IK,ATP was already activated. Conclusions: The response to levcromakalim in isolated cells from pulmonary artery was, as expected for an agent activating KATP channels, modulated by changes in the pipette [ATP]. This effect was not dependent on the presence of other pipette nucleotides, although the possibility cannot be excluded that metabolites from the cellular breakdown of ATP are essential for normal channel regulation. GDP could also activate IK,ATP under conditions where the channel is probably in a low phosphorylation state. The time-dependent effects of GTP require further work to determine the precise mechanism, but may suggest that GTP and/or G-proteins are involved in the regulation of KATP channels.