Adipocyte function is highly regulated in response to changing oxygen levels and physiological regulation of adipocyte formation involves factors originally identified as hypoxia-responsive proteins. Inhibition of adipogenesis at low oxygen tension is associated with activation of hypoxia inducible factor-1 alpha (HIF-1alpha), a transcription factor essential for cellular responses to decreased oxygen levels whose activity is regulated by prolyl hydroxylase (PHD) enzymes. However, HIF-1alpha RNA expression has been detected during the initial stages of adipocyte formation under aerobic conditions, suggesting a physiological role for HIF-1alpha during adipogenesis under a range of oxygen levels. Here we investigated the expression of HIF-1alpha during adipogenesis using the murine 3T3-L1 adipocyte model. Our results indicate the tissue-specific form of HIF-1alpha is upregulated during adipogenesis with maximal levels obtained within the first 24 h after induction. The increase in HIF-1alpha l.1 gene expression corresponds to increased nuclear HIF-1alpha protein, which gradually declines throughout adipogenesis under aerobic conditions. Each of the three HIF prolyl hydroxylases involved in regulating HIF-1alpha stability is expressed during adipogenesis. The prolyl hydroxylase domain 1 (PHD1) isoform of the HIF prolyl hydroxylases is expressed in early adipogenesis and the PHD2 and PHD3 isoforms are expressed during late adipogenesis. Pharmacological inhibition of PHD activity during the initial stages of adipogenesis abrogates the formation of adipocytes and inhibits gene expression of each of the PHD. However, inhibition of PHD activity does not consistently regulate HIF-1alpha l.1 expression or HIF-1alpha protein levels, suggesting that hydroxylation-independent mechanisms are involved in regulating HIF-1alpha expression in adipocytes under aerobic conditions.