BACKGROUND Satellite cell proliferation, reinnervation, and revascularization were studied in human nonreinnervated free microvascular muscle flaps to characterize mechanisms of muscle regeneration after flap surgery. METHODS Patient biopsies (n = 19) were taken at operation and five timepoints up to 9 months after operation, and corresponding clinical data were obtained. Immunohistochemistry for Ki-67 was used to detect proliferating satellite cells, CD-31 to identify endothelial cells, and S-100 and PGP 9.5 proteins to detect reinnervation. RESULTS Two weeks after operation, the expression of PGP 9.5 and S-100 had virtually disappeared in all larger nerve fibers and half of smaller nerve fibers. By 6 months, however, a strong expression of PGP 9.5 and S-100 had reappeared in larger nerve fibers in three of four flaps, suggesting that reinnervation had taken place. The number of mitotic satellite cells already peaked at 2 weeks, indicating onset of muscle regeneration. The number of intramuscular capillaries first increased but later decreased to lower than original level. Flaps with more muscle volume showed more reinnervation and satellite cell mitotic activity. In cases of a delay occurring in reconstructive surgery, a low level of reinnervation was seen. CONCLUSIONS Three patients of four showed spontaneous muscle reinnervation in microvascular free flaps with satellite cell activation followed by restored morphology. Late reconstruction and obesity lead to poor reinnervation, placing emphasis on timing of surgery and patient selection.