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 oxygen consumption (VO₂). Kinase phosphorylation in high-oxidative red (RTA), and low-oxidative white tibialis anterior (WTA) with 2.5-fold differences in mitochondrial content were compared. Stimulation of the TA muscle elicited large increases in VO₂ (3-6-fold above resting levels in WTA, 4-60-fold increases in RTA). At rest, AMPK, p38, p42, and p44 activation were ~2-fold greater in WTA, compared to 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 VO₂ for p38 and p42. During increases in VO₂ up to 6-fold above rest, greater responses were observed in RTA than WTA for AMPK and p44, while AKT activation was greater in WTA. In RTA, elevations in VO₂ elicited increases in AMPK and p44 activation, while AKT, p38, and p42 were less sensitive to increments in VO₂. ROS production was greater in mitochondria from white muscle, but when calculated in the context of the whole muscle, was 2-fold greater in red compared to 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 VO₂, suggesting that muscle mitochondrial content is important, but not the sole determinant of kinase activation during exercise of different intensities.