One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.