Activation of viral promoter transcription is a crucial event in the life cycle of several viruses. Hypoxia inducible factor-1 alpha (HIF-1alpha) is an inducible transcription factor whose activity is dependent on environmental conditions, most notably oxygen levels and cellular stress. HIF-1alpha has been implicated in the pathogenesis of several viruses, including HIV-1, HHV-8 and RSV. Under hypoxic conditions or oxidative stress, HIF-1alpha becomes stable and translocates to the nucleus, where it modulates gene transcription. The objective of the present study was to investigate a possible role for HIF-1alpha in the activation of JCV. Glial cell cultures infected with JCV demonstrated a significant increase in the levels of HIF-1alpha, in where it is located to the nucleus. Immunohistochemical studies corroborated upregulation of HIF-1alpha in JCV infected oligodendrocytes and astrocytes in clinical samples of PML compared with normal glial cells from the same samples in which HIF-1alpha expression is weak. CAT assays performed in co-transfected glial cells demonstrated activation of the JCV early promoter in the presence of HIF-1alpha. This activation was potentiated in the presence of Smad3 and Smad4. Finally, chromatin immunoprecipitation assays demonstrated the binding of HIF-1alpha to the JCV control region. These results suggest a role for HIF-1alpha in the activation of JCV; understanding of this pathway may lead to the development of more effective therapies for PML, thus far an incurable disease.