Physical exercise is a potent stimulator of mitogen-activated protein (MAP) kinase signaling. To determine if this activation is secondary to systemic responses to exercise or due to muscle contractile activity per se, an isolated muscle preparation was developed. Contractile activity in vitro significantly increased p44(MAPK) and p42(MAPK) phosphorylation by 2.9- and 2.4-fold, respectively. Contraction-stimulated MAP kinase phosphorylation was not decreased in the presence of D-tubocurarine or calphostin C, suggesting that neither neurotransmitter release nor diacylglycerol-sensitive protein kinase C mediates the contraction-induced activation of this signaling cascade. However, PD-98059, an inhibitor of MAP kinase kinase (MEK), inhibited the contraction-induced increases in MAP kinase phosphorylation. PD-98059 did not alter contraction-induced increases in glucose uptake or glycogen synthase activity, demonstrating that MAP kinase signaling is not necessary for these important metabolic effects of contractile activity in skeletal muscle. These data suggest that contractile activity of the skeletal muscle fibers per se, and not responses to neurotransmitter release, hormones, or other systemic factors, is responsible for the stimulation of MAP kinase signaling with physical exercise.