In most cell culture studies, cells are grown on smooth culture surfaces. Using microfabrication technology, we have developed microgrooved silicone surfaces to grow cells and subject them to repetitive mechanical stretching. When human patellar tendon fibroblasts were plated on these microgrooved surfaces, the cells had an elongated shape and underwent cyclic uniaxial stretching parallel to their long axes, all of which closely mimic conditions of tendon fibroblasts in vivo. Also, when fibroblasts were grown on microgrooves oriented at 45 and 90 degrees with respect to stretching direction, they did not change alignment or shape under cyclic mechanical stretching. Furthermore, compared to nonstretched cells, 8% cyclic stretching of tendon fibroblasts oriented at 0 (i.e., parallel to stretching direction), 45, and 90 degrees was found to increase α-SMA protein expression level by 46, 31, and 14%, respectively. In addition, 8% cyclic stretching tendon fibroblasts for 4 and 8 h oriented parallel to stretching direction increased α-SMA protein expression level by 25 and 57%, respectively. Thus, the results of this study showed that α-SMA protein expression levels of tendon fibroblasts depend on cell orientation with respect to stretching direction and stretching duration. We suggest that microgrooved silicone substrates can be used to study biological responses of tendon or ligament fibroblasts to repetitive mechanical stretching conditions in a more controlled manner.