Purpose: Strains tending to pull the periosteum away from the bone are typically osteogenic. The aim of this study was to assess the influence of periosteum on de novo bone formation in a rat calvaria model of distraction osteogenesis. Materials and Methods: A total of 28 rats were randomized in four experimental groups considering two treatment modalities. Periosteum was either left intact or completely excised. In half of the animals, the distraction plate was covered with a collagen membrane. All animals were subjected to a 7-day latency period and a 10-day distraction period. The samples were harvested after a 2-week consolidation period and analyzed histologically and by means of microcomputed tomography (micro-CT). Results: New bone in all animals originated from the original bone surface. Two groups of animals with periosteum, with membrane (24.56 ± 5.26) and without membrane (21.83 ± 14.04), showed significantly more bone volume compared with groups without periosteum, with membrane (2.72 ± 1.08, P = .003) and without membrane (4.25 ± 2.33, P = .014). There were no significant differences between the four groups in bone mineral density. Groups pooled together for the presence of periosteum demonstrated significantly more bone volume (P .001) and bone mineral density (P = .028) than groups without periosteum. No differences were found for groups pooled for the barrier membrane application. Conclusion: The periosteum plays an indispensable, but indirect role in the osteogenic process during periosteal distraction osteogenesis. Keywords: animal model, collagen membrane, distraction osteogenesis, periosteum