Our objective in work presented here was to understand the mechanisms by which activated p38α MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38α MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38α MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of α-tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38α MAPK hearts. Our experiments also showed that p38α MAPK colocalizes with α-actinin at the Z-disc and complexes with protein phosphatases (PP2α, PP2β). These data are the first to indicate that chronic activation of p38α MAPK directly depresses sarcomeric function in association with decreased phosphorylation of α-tropomyosin.