Hypoxia-inducible factor-1 (HIF-1) is a key regulator of tumor cell hypoxia. It regulates the expression of several genes related to oxygen homeostasis in response to hypoxic stress. Carbonic anhydrase IX (Ca-IX) has been found to be a stable marker of acute or chronic hypoxia. N-Myc down-regulated gene 1 (NDRG1) has been shown to possess more specific characteristics for clinical analysis and identification purposes. HIF-1 activates gene expression of the two genes and promotes tumor cell survival under hypoxic conditions. Herein, we modified a flow cytometry protocol to separate NDRG1- and CA-IX-negative and -positive cells in vitro to sort chronically hypoxic cells from glioblastoma tumors. The FITC-anti-CA-IX fluorescence differed between positive and negative cells by a factor of 60-160 in U373, U87-MG, U251 and GaMG, respectively. A clear effect of the O2 concentration on CA-IX expression was visible in GaMG and U251 cell lines whereas U373 showed a less differentiated pattern. NDRG1 expression was present in U373, U251 and GaMG with the lowest expression rate in GaMG. It was stable over 48 h of reoxygenation after 24 h of extreme hypoxia (0.1% O2). During reoxygenation NDRG1 was relatively stable in the four tumor cell lines with the lowest expression in GaMG. An oxygen- and time-dependent elevation of nuclear HIF-1alpha binding on HRE was displayed. FACS analysis of CA-IX and NDRG1 expression may be a new approach to determining the hypoxic state of tumor cells. However, an extensive analysis of other hypoxia-regulated genes in different tumors is required to identify additional markers for the detection of the oxygenation state in human tumors in order to tailor effective tumor-specific therapeutic strategies.