Thrombospondin-l (TSP-1) is believed to be an endogenous angiogenic inhibitor. In this study, we report that a single 1 h bout of treadmill running increases TSP-1 mRNA 3-4-fold (p < 0.001). Interestingly, with short-term training (up to 5 days, 1 h/day) the acute response of TSP-1 mRNA to exercise was ablated after 3 days. Following long-term training (8 weeks, 1 h/day, 5 d/wk), in either normoxia or chronic hypoxia, the TSP-1 mRNA response to an acute bout of exercise was restored and increased 3-4-fold (p < 0.01). However, chronic exposure to hypoxia (8-weeks) decreases both the basal and acute exercise-induced TSP-1 mRNA levels by 44 and 48%, respectively (p < 0.05). Based on the robust TSP-1 gene response to a single acute exercise bout, its temporal response to repetitive exercise bouts, and the putative role of TSP-1 in the angiogenic process, we speculate that TSP-1 may play a role in regulating the onset of skeletal muscle angiogenesis in response to exercise.