Hypoxia inhibits alveolar ion transport, a process that is mimicked by ROS-scavengers but prevented by H_2O_2. However, results on ROS production in hypoxia and ROS-involvement in signalling are divergent. We measured ROS-formation during 15 min (37°C) exposure of lung alveolar A549 cells to hypoxia (2% O2), normoxia (21% O2) and hyperoxia (35% O2) by chemiluminescence (CL), ESR-spectroscopy and DCF-fluorescence, and studied whether changes in the NFkB-system, known to be involved in ROS-dependent cellular signalling is affected. CL and ESR indicate a decreased accumulation of ROS in hypoxia and an increased ROS-formation at higher oxygen. In contrast, DCF-fluorescence was increased in hypoxia. In all experiments, values became normal upon return to 21% O2. N-acetyl-cysteine decreased CL in all states of oxygenation but the O2-dependency persisted. Addition of rotenone and antimycin A to inhibit the respiratory chain increased ROS-formation, whereas stimulation of the complexes I and II by increasing ADP decreased ROS formation measured by CL. In the ADP-stimulated state, CL was lower in hypoxia than normoxia. NFkB measured by EMSA did not change significantly. Our results confirm apparent discrepancies on ROS in hypoxia indicating that these might be explained on the basis of different species of ROS seen by the different detection methods. The changes in ROS observed under these conditions seem not to affect NFkB-related signalling.