Abstract The hypoxia-inducible factor 1 (HIF-1) transcription factor is a critical mediator of the cellular response to oxygen deprivation. HIF-1α is frequently overexpressed in human cancers due to intratumoral hypoxia as well as genetic alterations that promote HIF-1α synthesis or inhibit its degradation (i.e. gain of function mutations in oncogenic pathways, such as RAS and PI3K, or loss of function mutations to the tumor suppressor, VHL, respectively). Under hypoxic conditions, HIF-1α is stabilized and translocates to the nucleus where it activates the transcription of genes that promote angiogenesis, cell proliferation/survival, and metastasis, among other processes. While the role of HIF-1 in the regulation of the cell cycle has been well studied, the effect of the cell cycle stage on HIF-1 expression and activity remains unknown. To address this question, HCT116 colon cancer cells were synchronized in G1, S, or G2/M-phase and exposed them to hypoxia (0.2% O2). Interestingly, HIF-1α expression was nearly abolished upon G2/M-phase arrest, and a significant increase was observed following arrest in S-phase. Therefore, cell cycle position influences the expression of HIF-1α, independent of oxygen concentration. Previous data in our lab indicates that knockdown of CDK1 decreased HIF-1α expression under hypoxic conditions. To further characterize this effect, we demonstrate that acute inhibition of CDK1 kinase activity with small molecules significantly decreases the stability of HIF-1α at the protein level, without affecting HIF-1α mRNA levels. Furthermore, cycloheximide chase experiments reveled that HIF-1α protein levels decreased at a significantly faster rate in the presence of a CDK1 inhibitor, R0-3306, (t1/2 = 38.2 ± 6.1 min) compared to controls (t1/2 = 54.6 ± 3.2 min). These results indicate that the half-life of HIF-1α is significantly decreased upon inhibition of CDK1. Taken together, these data indicate that CDK1 activity regulates the stability of HIF-1α, and suggests that CDK1 activity stabilizes HIF-1α through a post-translational modification that promotes its stability. Thus, we have discovered that CDK1 and the cell cycle position regulate the expression of HIF-1α. There is a subset of tumors that express HIF-1α under normoxic conditions, driving tumor progression and therapeutic resistance. Therefore the identification of alternative mechanisms that regulate HIF-1α expression would be extremely valuable in the clinical setting, as well as to aid the basic understanding of the role of HIF-1 in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-484. doi:1538-7445.AM2012-LB-484