Tumor hypoxia and the hypoxia-inducible factors (HIFs) play a central role in the development of cancer. To study the relationship between tumor growth, tumor hypoxia, the stabilization of HIF-1alpha, and HIF transcriptional activity, we have established an in vivo imaging tool that allows longitudinal and noninvasive monitoring of these processes in a mouse C51 allograft tumor model. We used positron emission tomography (PET) with the hypoxia-sensitive tracer [(18)F]-fluoromisonidazole (FMISO) to measure tumor hypoxia over 14 days. Stabilization of HIF-1alpha and HIF transcriptional activity were assessed by bioluminescence imaging using the reporter constructs HIF-1alpha-luciferase and hypoxia response element-luciferase, respectively, stably expressed in C51 cells. Interestingly, we did not observe any major change in the level of tumor hypoxia throughout the observation period whereas HIF-1alpha levels and HIF activity showed drastic temporal variations. When comparing the readouts as a function of time we found a good correlation between HIF-1alpha levels and HIF activity. In contrast, there was no significant correlation between the [(18)F]-FMISO PET and HIF readouts. The tool developed in this work allows for the longitudinal study of tumor hypoxia and HIF-1alpha in cancer in an individual animal and will be of value when monitoring the efficacy of therapeutical interventions targeting the HIF pathway.