Physical exercise and contraction increase c-Jun NH ₂-terminal kinase (JNK) activity in rat and human skeletal muscle, and eccentric contractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects of isometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus (EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats were subjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20-25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretch had a much more profound effect on the stress-activated protein kinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation of JNK occurred within the muscle fibers. These studies suggest that the stretch component of a muscle contraction may be a major contributor to the increases in JNK activity and p38 phosphorylation observed after exercise in vivo.