Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n=16) or without (n=10) 3 mM caffeine or during electrical stimulation (n=14). For each condition, rats were subdiveded and treated with either 10 µM KN92 or KN93, inactive and active CaMKII inhibitors respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation (P<0.05). KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P<0.05). Caffeine had no effect on ERK1/2 phosphorylation (P>0.05) and increased ₂-AMPK activity by 68% (P<0.05). Electrical stimulation increased ERK1/2 phosphorylation and ₂-AMPK activity by 51% and 3.4-fold respectively (P<0.05). KN93 had no effect on caffeine-induced ₂-AMPK activity or ERK1/2 phosphorylation or contraction-induced ERK1/2 phosphorylation (P>0.05). Alternatively, it decreased contraction-induced ₂-AMPK activity by 51% (P<0.05) suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca²⁺-independent activation of ERK1/2 as well as Ca²⁺-dependent activation of CaMKII and AMPK.