The sensitivity of different parameters for the determination of lung injury caused by nitrogen dioxide (NO2) was investigated. Male rats were exposed to concentrations of 0, 4, 10 or 25 ppm NO2 for 6 h/day, for 7, 14 or 21 days. Histopathology of the nasal cavity, larynx, trachea and lungs was compared with the changes in macrophage function and morphology. In addition several biochemical parameters were determined in lung lavages. Cytotoxic effects were investigated in primary cultures of rat and bovine alveolar macrophages, exposed to the same NO2-levels as in the in vivo exposure. Treatment-related histopathological changes were observed in the lungs. No differences between exposed and control animals were observed in the nasal cavity, larynx or trachea. The morphology of the lavaged alveolar macrophages was changed at all exposure concentrations on day 7, 14 and 21. An increase in the number of macrophages was found after exposure to 10 and 25 ppm NO2 on days 7, 14 and 21. The phagocytic capacity was diminished after 14 and 21 days exposure to 25 ppm and at both times exposure to 10 and 25 ppm increased the level of gamma-glutamyl transferase (GGT) in lavage fluids. Morphology of the macrophages and levels of GGT were found to be sensitive parameters of nitrogen dioxide toxicity. In vitro exposure of rat and bovine alveolar macrophages to comparable NO2-concentrations induced effects on phagocytosis similar to those observed for macrophages from exposed rats.