Muscle contractions strongly activate p38 MAP kinases, but the precise contraction-associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N-benzyl-p-toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze-clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non-stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction-induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways. J. Cell. Biochem. 114: 1445–1455, 2013. © 2013 Wiley Periodicals, Inc.