We studied the expression of cytosolic phospholipase-A2 and activity of secretory phospholipase-A2 by human patellar tendon fibroblasts subjected to cyclic mechanical stretching. The effect of different stretching frequencies on the production of prostaglandin-E2 and expression of cyclooxygenase enzyme were also examined. An in vitro system that can control alignment, shape, and mechanical loading conditions of tendon fibroblasts was used for this study. Cyclic stretching of fibroblasts increased the expression level of cytosolic phospholipase-A2 by 88% and activity level of secretory phospholipase-A2 by 190%, compared with those of nonstretched fibroblasts. Cyclic stretching of tendon fibroblasts at 0.1 Hz and 1.0 Hz also increased prostaglandin-E2 production by 40% and 69%, respectively. Furthermore, cyclooxygenase-1 and cyclooxygenase-2 expression levels were increased in a stretching frequency-dependent manner, but cyclooxygenase-2 expression was increased more than that of cyclooxygenase-1. Because cytosolic phospholipase-A2 and secretory phospholipase-A2 are involved in the production of prostaglandin-E2 and other inflammatory mediators, this study suggests that regulation of phospholipase-A2 expression level may be an alternative approach to control in vivo tendon inflammation. The results of this study also may explain in part why activities that involve repetitive motion and high frequency loading of tendons are more likely to result in tendon inflammation.