BACKGROUND: Defects of the adult skull do not heal spontaneously, producing challenging problems for the craniofacial surgeon. Reconstruction of such defects requires either the placement of alloplastic material or the harvest of autogenous bone. A technique is described for the reconstruction of critical-sized, full-thickness calvarial defects in the adult rat model using specific adult stem cells, namely, multipotent adult stem cells. METHODS: The cells were harvested from adult skeletal muscle and cultured in an undifferentiated state within a matrix of polyglycolic acid mesh. An 8-mm critical-sized defect was created in the calvaria of adult rats and either left empty, filled with polyglycolic acid mesh alone, or filled with multipotent adult stem cells seeded into the polyglycolic acid mesh. After 12 weeks, the calvariae were harvested, stained, and blind graded by light microscopy on the presence or absence of reconstituted bone. RESULTS: A total of 22 animals were available for study: seven from the empty defect group, eight from the polymer group, and seven from the polymer plus stem cell group. The mean scores for the three groups were 1.9, 2.3, and 5.3, respectively. Statistical analysis showed statistical significance among the groups as a whole (p < 0.01) and between the polymer plus stem cell group and the empty defect and polymer-alone group. CONCLUSIONS: The results demonstrate that regeneration of calvarial bone is possible using stem cells harvested from adult skeletal muscle and seeded into a polyglycolic matrix. The technique may ultimately be used in clinical practice to reconstruct calvarial defects.