Acute contractile activity increases the activation of protein kinases involved in signal transduction. We hypothesized that the contractile activity-induced kinase phosphorylation would occur to a lesser degree in muscle with elevated mitochondrial content. We compared red and white sections of tibialis anterior (TA) muscle with two- to threefold 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, 3 h/day). Both the chronically stimulated and the contralateral control muscles were then acutely stimulated in situ for 15 min (10 Hz). We investigated 1) the total protein content and 2) the phosphorylation of kinases important for mitochondrial biogenesis in skeletal muscle, including AMPKα and 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 phosphorylation 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 phosphorylation in muscle with high mitochondrial content suggests that these proteins may become less sensitive to upstream signaling and require greater stimulation for activation to propagate these adaptive cues downstream toward transcription or translation events.