The healing after rotator cuff surgery is still dissatisfying, and increased muscle fatty infiltration even more impairs the healing success. To achieve sufficient healing after rotator cuff reconstructions, the use of growth factors may be one possibility. The aim of the study was to identify a possible relationship between fatty infiltration of the supraspinatus muscle and cellular biological characteristics and stimulation potential of tenocyte-like cells (TLCs). TLCs of 3 donor groups differing in grade of muscle fatty infiltration were analyzed for their cellular characteristics and were stimulated with BMP-2 or BMP-7 in a 3D scaffold culture. The cell count and potency for self-renewal were significantly decreased in TLCs from donors with high muscle fatty infiltration compared to the lower fatty infiltration groups. Cell count and collagen-I expression as well as protein synthesis were stimulated by growth factors. Interestingly, TLCs of the high fatty infiltration group exhibited a weaker stimulation potential compared to the other groups. TLCs from donors with high muscle fatty infiltration generally revealed inferior characteristics compared to cells of lower fatty infiltration groups, which may be one reason for a weaker healing potential and may represent a possible starting point for the development of future treatment options. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:129–137, 2014.