BACKGROUND: Recent studies report a relatively high failure rate for tendon-bone healing after rotator cuff repair. Several studies have investigated biologically augmented rotator cuff repair; however, none has shown the application of synovial mesenchymal stem cells for such repair. PURPOSE: To demonstrate whether cells derived from shoulder tissues have mesenchymal stem cell properties and to identify which tissue is the best source of the mesenchymal stem cells. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-two patients with a diagnosed rotator cuff tear preoperatively were enrolled in this study. Human mesenchymal tissues were obtained during arthroscopic surgery for rotator cuff tears from 19 donors who met the inclusion criteria and had investigable amounts of tissue. Colony-forming units, yield obtained, expandability, differentiation potential, epitope profile, and gene expression were compared among the cells from 4 shoulder tissues: synovium of the glenohumeral joint, subacromial bursa, margin of the ruptured supraspinatus tendon, and residual tendon stump on the greater tuberosity (enthesis). RESULTS: The number of live passage 0 cells from whole tissue was significantly higher in cells derived from the subacromial bursa (P < .05). Subacromial bursa-derived cells retained their expandability even at passage 10. In adipogenesis experiments, the frequency of Oil Red O-positive colonies was significantly higher for synovium- and subacromial bursa-derived cells than for tendon- and enthesis-derived cells (P < .0001). In studies of osteogenesis, the rate of von Kossa- and alkaline phosphatase-positive colonies was highest in subacromial bursa-derived cells (P < .0001). The chondrogenic potential was highest in cells derived from the enthesis. For epitope profiling, 11 surface antigens were measured, and most had similar epitope profiles, irrespective of cell source. CONCLUSION: The findings indicate that the subacromial bursa is a good candidate for the source of mesenchymal stem cells in rotator cuff tears. CLINICAL RELEVANCE: Synovial cells from the subacromial bursa in patients with rotator cuff tears are a superior cell source in vitro, suggesting that mesenchymal stem cells from this tissue could be good candidates for biological augmentation of rotator cuff repair.