Objectives The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1alpha (HIF1alpha) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1alpha has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood. Methods We investigated the conditions for normoxic HIF1alpha stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR. Results Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1alpha in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1alpha. Additionally, the normoxic HIF1alpha protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1alpha was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1alpha to the same extent as glutamine. Conclusion Our study suggests that HIF1alpha is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1alpha and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions. Clinical relevance Therefore, the inhibition of HIF1alpha (and/or HIF1alpha target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.