As a central component of a novel protein kinase cascade, the activation of the mitogen-activated protein (MAP) kinase cascade has attracted considerable attention. We sought to determine the effect of exercise and diet on the activation of the extracellular-signal regulated protein kinase (ERK) 1/2 and the p38 MAP kinase pathways in rat soleus muscle. Forty-eight Sprague-Dawley rats were assigned to one of two dietary conditions: high-carbohydrate (CHO) or high-fat (FAT). Animals having each dietary condition were further divided into one of three subgroups: a sedentary control group that did not exercise (NT), a group that performed 8 weeks of treadmill running and was sacrificed 48 h after their final treadmill run (CE), and a group that was sacrificed immediately after their final routine exercise training (AE). A high-fat diet did not have any significant effect on phosphorylated and total forms of ERK 1/2 or p38 MAP kinase. In chronically trained muscle that was taken 48 h after the last training, phosphorylated ERK 1/2 significantly increased only in the FAT but not in the CHO groups. In the case of total ERK 1/2, it increased significantly for both groups. In contrast, both phosphorylated and total forms of p38 MAP kinase decreased markedly compared to sedentary muscle. In muscle that was taken immediately after a last bout of exercise, phosphorylated ERK 1/2 increased in both groups but statistical significance was seen only in the CHO group. Total ERK 1/2 in acutely stimulated muscle increased only in the CHO-AE group even though the degree was much lower than the phosphorylated status. Muscle that was taken immediately after the routine training increased in phosphorylation status of p38 MAP kinase for both dietary conditions. However, statistical significance was seen only in the CHO group owing to a large variation with FAT. In conclusion, a high-fat diet per se did not have any notable effect versus a high-carbohydrate diet on MAP kinase pathways. However, when diet (either CHO or FAT) was combined with exercise and/or training, there was differentiated protein expression in MAP kinase pathways. This indicates MAP kinase pathways have diverse control mechanisms in slow-twitch fibers.