Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long-term production of prostaglandin F2 alpha, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P < .005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch-activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's Km, with little change in its Vmax. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen-inducible isoform of cyclooxygenase (PGHS-2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch-induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch-induced cyclooxygenase activation. Pertussis toxin also inhibited stretch-induced increases in PGF2 alpha production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein-dependent process involving the activation of cyclooxygenase, an immediate early gene product.