Expression patterns of the three isoforms of the regulatory γ-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The γ3-isoform appeared highly specific to skeletal muscle, whereas γ1 and γ2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of γ3. In samples of white skeletal muscle, γ3 clearly appeared to be the most abundant γ-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of γ3 mRNA expression, whereas levels of γ1 and γ2 remained largely unchanged. However, even in these cultured myotubes, γ2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a γ3-specific antibody showed that γ3 was exclusively associated with the α2- and β2-subunit isoforms. The observation that the AMPKγ3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant γ-isoform, strongly suggests that γ3 has a key role in this tissue.