Early placentation occurs in a relatively hypoxic environment. As placentation progresses, the events associated with trophoblast differentiation in response to increasing oxygen are known; however, the mechanisms by which trophoblast cells 'sense' the changing oxygen tension remain unclear. HIF-1alpha mediates oxygen's effects on trophoblast differentiation. VHL has been associated with negative regulation of HIF-1alpha under oxygenated conditions. The aim of this study was to elucidate mechanisms of oxygen sensing in trophoblast cells. Mitochondrial electron transport chain interference in first trimester villous explants cultured under low oxygen inhibited extravillous trophoblast cell outgrowth associated with reduced cellular proliferation. HIF-1alpha immunoreactivity was decreased. VHL displayed a similar pattern of expression as HIF-1alpha in the first trimester and in response to varying oxygen; however, spatially they showed reciprocal regulation. Together, these findings suggest a role for the mitochondrial pathway in trophoblast cell oxygen sensing, and a role for VHL in sensing, potentially via an interaction with HIF-1alpha.