ATP-sensitive potassium (KATP) channels regulate insulin secretion, vascular tone, heart rate and neuronal excitability by responding to transmitters as well as the internal metabolic state. KATP channels are composed of four pore-forming α-subunits (Kir6.2) and four regulatory β-subunits, the sulfonylurea receptor (SUR1, SUR2A or SUR2B). Whereas protein kinase A (PKA) phosphorylation of serine 372 of Kir6.2 has been shown biochemically by others, we found that the phosphorylation of T224 rather than S372 of Kir6.2 underlies the catalytic subunits of PKA (c-PKA)- and the D1 dopamine receptor-mediated stimulation of KATP channels expressed in HEK293 cells. Specific changes in the kinetic properties of channels treated with c-PKA, as revealed by single-channel analysis, were mimicked by aspartate substitution of T224. The T224D mutation also reduced the sensitivity to ATP inhibition. Alteration of channel gating and a decrease in the apparent affinity for ATP inhibition thus underlie the positive regulation of KATP channels by PKA phosphorylation of T224 in Kir6.2, which may represent a general mechanism for KATP channel regulation in different tissues.