Angiotensin II (AngII) inhibits bTREK-1 K⁺ channels in bovine adrenocortical cells through a Gq-coupled AT₁ receptor by activation of separate Ca²⁺- and ATP hydrolysis-dependent signaling pathways. Whole-cell patch clamp recording from bovine adrenal zona fasciculata (AZF) cells was used to characterize the ATP-dependent signaling mechanism for inhibition of bTREK-1 by AngII. We discovered that ATP-dependent inhibition of bTREK-1 by AngII occurred through a novel mechanism that was independent of phospholipase C (PLC) and its downstream effectors. The ATP-dependent inhibition of bTREK-1 by AngII was not reduced by the PLC antagonists edelfosine and U73122, or by the PKC antagonists bis-indoyl-maleimide (BIM) or calphostin C. Addition of DiC₈PI(4,5)P₂, a water-soluble derivative of phosphotidyl inositol 4,5 bisphosphate (PIP₂) to the pipette solution failed to alter inhibition by AngII. bTREK-1 inhibition by AngII was also insensitive to antagonists of other protein kinases activated by AngII in adrenocortical cells, but was completely blocked by inorganic polytriphosphate PPPi. DiC₈PI(4,5)P₂ was a weak activator of bTREK-1 channels, when compared to the high affinity ATP analog N⁶-(2-Phenylethyl)adenosine-5'-O-triphosphate (6-PhEt-ATP). These results demonstrate that the modulation of bTREK-1 channels in bovine AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors. AngII inhibits bTREK-1 channels through a novel pathway that is different from that described for inhibition of TREK-1 channels in neurons, or cloned channels expressed in cell lines. They also indicate that AngII inhibits bTREK-1 and depolarizes AZF cells by two, novel, independent pathways that diverge proximal to the activation of PLC.