The AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. The gamma subunit is essential for enzyme activity by virtue of its binding to the C-terminus of the alpha subunit and appears to play some role in the determination of AMP sensitivity. We demonstrate that a gamma1R70Q mutation causes a marked increase in AMPK activity and renders it largely AMP-independent. This activation is associated with increased phosphorylation of the alpha 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 gamma1 subunit in the regulation of AMPK and its modulation by AMP.