1. Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen-activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise-related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. 2. Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal-regulated kinase 1 and 2 (MAPK(erk1/2)) and stress-activated protein kinase p38 (MAPK(p38)). 3. Concentric contractions induced a 5-fold increase in MAPK(erk1/2) phosphorylation. Application of the antioxidants N-acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction-induced increase in MAPK(erk1/2) phosphorylation. Mild passive stretches of the muscle increased MAPK(erk1/2) phosphorylation by 1.8-fold, whereas the combination of acidosis and passive stretches resulted in a 2.8-fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPK(p38). 4. High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7- and 9.5-fold increases of phosphorylated MAPK(erk1/2), respectively, whereas phosphorylation of MAPK(p38) increased by 7.6- and 1.9-fold (not significant), respectively. 5. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPK(erk1/2) and MAPK(p38) is induced by mechanical alterations, whereas contraction-related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPK(erk1/2) only. Thus, contraction-induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPK(erk1/2) and by high mechanical stress for MAPK(p38).