Acellular matrix obtained from homologous muscular tissue has been previously used to repair muscular defects. However, the implants, although not rejected, give rise to an intense inflammatory response and are rapidly replaced by fibrous tissue. In this study we examined the possibility that co-culture with autologous satellite cells can improve the efficiency of homologous acellular matrix as skeletal muscle substitute. Satellite cells, isolated from rat dorsal muscle, were cultured in vitro on homologous acellular matrix obtained by detergent-enzymatic treatment of abdominal muscle fragments. Scanning electron microscopy revealed that after 24 h of co-culture satellite cells were attached to the matrix, but still possessed a round shape. After 96 h, seeded cells began to flatten and to differentiate, originating few multinucleated myotubes. Patches of homologous matrix, seeded or not with autologous satellite cells, were implanted in the dorsal muscle of rats. At autopsy, the implants were recovered and processed for light microscopy. Two weeks after surgery, fibrous tissue started to replace the grafts composed only by acellular matrix, which at the 4th week were transformed into a fibrous scar. In contrast, at both times post-surgery the structure of implants containing autologous satellite cells was well preserved. The inflammatory reaction was modest and fibrosis was confined to the periphery of the grafts. It is concluded that the presence of autologous satellite cells is an important factor to preserve the structural integrity and to improve in vivo biocompatibility of homologous muscular acellular matrix implants.