The AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic α subunit and two regulatory subunits, β and γ. The γ subunit is essential for enzyme activity by virtue of its binding to the C-terminus of the α subunit and appears to play some role in the determination of AMP sensitivity. We demonstrate that a γ1R70Q mutation causes a marked increase in AMPK activity and renders it largely AMP-independent. This activation is associated with increased phosphorylation of the α subunit activation loop T172. These in vitro characteristics of AMPK are also reflected in increased intracellular phosphorylation of one of its major substrates, acetyl-CoA carboxylase. These data illustrate the importance of the γ1 subunit in the regulation of AMPK and its modulation by AMP.