Acute contractile activity increases the activation of protein kinases involved in signal transduction. We hypothesized that the contractile activity-induced kinase activation would occur to a lesser degree in muscle with elevated mitochondrial content. We compared red and white sections of tibialis anterior (TA) muscle with 2-3 fold differences in mitochondrial volume, and we increased the mitochondrial content in the TA muscle by 40% with unilateral chronic stimulation-induced contractile activity (10 Hz, 7 days, 3hr/day). The chronically simulated and the contralateral control muscles were then both acutely stimulated in situ for 15 mins (10 Hz). We investigated 1) the total protein content, and 2) the phosphorylation of kinases important for mitochondrial biogenesis in skeletal muscle, including AMPK, p44, p42, and p38 MAPKs, as well as AKT by immunoblotting. In response to chronic stimulation, a selective upregulation of kinase protein content was observed, suggesting unique transcriptional/translational processing for these enzymes. Inverse relationships were observed between mitochondrial volume and 1) kinase protein content, and 2) basal levels of kinase phosphorylation. In general, the kinase activation response to acute exercise depended, in part, on the oxidative capacity of the fiber type, evidenced by a greater absolute level of acute contractile activity-induced kinase signaling in muscle with a lower mitochondrial volume. The attenuation of contraction-evoked kinase activation in muscle with high mitochondrial content suggests that these proteins may become less sensitive to upstream signaling, and require greater stimulation for activation in order to propagate these adaptive cues downstream, toward transcription or translation events.