Activation of AMP-activated protein kinase (AMPK) by exercise and metformin is beneficial for the treatment of type 2 diabetes. We recently found that, in cultured cells, the LKB1 tumor suppressor protein kinase activates AMPK in response to the metformin analog phenformin and the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have also reported that LKB1 activates 11 other AMPK-related kinases. The activity of LKB1 or the AMPK-related kinases has not previously been studied in a tissue with physiological relevance to diabetes. In this study, we have investigated whether contraction, phenformin, and AICAR influence LKB1 and AMPK-related kinase activity in rat skeletal muscle. Contraction in situ, induced via sciatic nerve stimulation, significantly increased AMPKalpha2 activity and phosphorylation in multiple muscle fiber types without affecting LKB1 activity. Treatment of isolated skeletal muscle with phenformin or AICAR stimulated the phosphorylation and activation of AMPKalpha1 and AMPKalpha2 without altering LKB1 activity. Contraction, phenformin, or AICAR did not significantly increase activities or expression of the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 in skeletal muscle. The results of this study suggest that muscle contraction, phenformin, or AICAR activates AMPK by a mechanism that does not involve direct activation of LKB1. They also suggest that the effects of excercise, phenformin, and AICAR on metabolic processes in muscle may be mediated through activation of AMPK rather than activation of LKB1 or the AMPK-related kinases.