Studies in nonmuscle cells have demonstrated that Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream regulators of AMP-activated protein kinase (AMPK) and Akt. In skeletal muscle, activation of AMPK and Akt has been implicated in the regulation of glucose uptake. The objective of this study was to determine whether CaMKKalpha regulates skeletal muscle glucose uptake, and whether it is dependent on AMPK and/or Akt activation. Expression vectors containing constitutively active CaMKKalpha (caCaMKKalpha) or empty vector were transfected into mouse muscles by in vivo electroporation. After 2 weeks, caCaMKKalpha was robustly expressed and increased CaMKI (Thr(177/180)) phosphorylation, a known CaMKK substrate. In muscles from wild-type mice, caCaMKKalpha increased in vivo [(3)H]-2-deoxyglucose uptake 2.5-fold and AMPKalpha1 and -alpha2 activities 2.5-fold. However, in muscles from AMPKalpha2 inactive mice (AMPKalpha2i), caCaMKKalpha did not increase AMPKalpha1 or -alpha2 activities, but it did increase glucose uptake 2.5-fold, demonstrating that caCaMKKalpha stimulates glucose uptake independent of AMPK. Akt (Thr(308)) phosphorylation was not altered by CaMKKalpha, and caCaMKKalpha plus insulin stimulation did not increase the insulin-induced phosphorylation of Akt (Thr(308)). These results suggest that caCaMKKalpha stimulates glucose uptake via insulin-independent signaling mechanisms. To assess the role of CaMKK in contraction-stimulated glucose uptake, isolated muscles were treated with or without the CaMKK inhibitor STO-609 and then electrically stimulated to contract. Contraction increased glucose uptake 3.5-fold in muscles from both wild-type and AMPKalpha2i mice, but STO-609 significantly decreased glucose uptake (approximately 24%) only in AMPKalpha2i mice. Collectively, these results implicate CaMKKalpha in the regulation of skeletal muscle glucose uptake independent of AMPK and Akt activation.