Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance transporter P-glycoprotein (P-gp) has not been investigated. Herein, we demonstrate that with increasing size of DU-145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the alpha-subunit of HIF-1 (HIF-1alpha) and P-gp were up-regulated. Furthermore, P-gp was up-regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro-oxidants H2O2 and buthionine sulfoximine down-regulated HIF-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha and P-gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF-1beta. Chemical hypoxia robustly increased HIF-1alpha as well as P-gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P-gp in multicellular tumor spheroids is under the control of HIF-1.