Muscle contractions activate protein kinases, leading to signal transduction. We hypothesized that kinase activation would be influenced by mitochondrial content, as well as by contractile activity-induced increases in muscle O2 consumption (V̇o2). Kinase phosphorylation in high-oxidative red and low-oxidative white tibialis anterior (TA) muscle (RTA and WTA, respectively) with 2.5-fold differences in mitochondrial content were compared. Stimulation of the TA muscle elicited large increases in V̇o2 (3- to 6-fold and 4- to 60-fold above resting levels in WTA and RTA, respectively). At rest, AMP-activated protein kinase (AMPK), p38, p42, and p44 activation were nearly twofold greater in WTA than in RTA, suggesting an inverse relationship between mitochondrial content and kinase activation in resting muscle. During contractions, similar degrees of phosphorylation in RTA and WTA were evident as a function of V̇o2 for p38 and p42. During increases in V̇o2 up to sixfold above rest, greater responses were observed in RTA than in WTA for AMPK and p44, whereas Akt activation was greater in WTA. In RTA, elevations in V̇o2 elicited increases in AMPK and p44 activation, whereas Akt, p38, and p42 were less sensitive to increments in V̇o2. Reactive oxygen species (ROS) production was greater in mitochondria from white muscle, but when it was calculated in the context of the whole muscle, ROS production was twofold greater in red than in white myofibers. Thus mitochondrial content influences ROS production and is inversely related to kinase activation in resting muscle. During contractions, kinases are differentially sensitive to contraction-induced increments in V̇o2, suggesting that muscle mitochondrial content is important, but it is not the sole determinant of kinase activation during exercise of different intensities.