Background: Transport distraction osteogenesis (DO) can be used to autologously reconstitute calvarial defects. However, distraction gap biology in transport DO has not been adequately described. The purpose of this study is to histomorphologically interrogate osteogenic formation during cranial transport distraction using a novel device. We also evaluate the effect of fat grafting on the regenerate during distraction. Methods: This study was approved by Yale IACUC (# 2011-11393). Ten male New Zealand white rabbits (3 months; 3.5kg) were used (8 treatment, 2 control). A 16x16mm defect was created abutted by a 10x16mm transport disc. The device was fixated anterioposteriorly. Four animals were fat-grafted using 2cc of subdermal intrascapular fat deposited along the distraction site. Latency (1d), active distraction (12-14d) (1.5 mm/day), and consolidation (4wks) followed. Calcein and xylene orange fluorochromes were injected subcutaneously during and post-distraction to mark sites of bone formation. Following sacrifice, osteogenesis was assessed using microCT, histology, and fluorescence. Results: No perioperative complications were experienced. Treatment animals demonstrated regenerate bone between distracted segments on microCT. MicroCT analysis of fat-grafted and non-fat grafted animals revealed a mean density of 2271.95 mgHA/ccm and 2254.27 mgHA/ccm (p=0.967), respectively, and defect bone versus total volume (BV/TV) of 0.0999 and 0.0766 (p=0.5979), respectively. Controls had minimal reossification. Histologically, mean densities measured 43.63% and 8.19% for non-fat and fat grafted animals, respectively. Density ratios (regenerate:native bone) were 53.96% and 23.71%, respectively. Fluorescent microscopy revealed ossification from the callus as well as bone fronts emanating from dura and periosteum. Conclusions: Transport distraction is effective to reconstruct critically-sized rabbit calvarial defects. Regenerate bone arises predominantly from the callus with contribution from surrounding dura and periosteum. Adipose grafting is well tolerated but does not enhance osseous regeneration. Keywords: Distractionosteogenesis, rabbit, in vivo, histology, fat transfer