In acute lung injury, a disturbed surfactant system may impair gas exchange. Previous evaluations of hyperoxia effects on surfactant proteins (SPs) followed exposures >1-2 days. To evaluate the effects of brief exposure to hyperoxia on the SP system, we exposed adult male rats to 95% O2 or air for 12, 36, and 60 h. SP-A, -B, and -C mRNAs were analyzed by Northern blot and semiquantitative in situ hybridization (ISH). SP-A and -B were analyzed in whole lung homogenates, lung lavage fluid, and fixed tissue by semiquantitative immunohistochemistry (IHC). All SP mRNAs were diminished at 12 h and rose to or exceeded control by 60 h as determined by Northern blot and ISH. These effects were seen mainly in the intensity of ISH signal per cell in both type II and bronchiolar epithelial (Clara) cells and to a lesser extent on numbers of positively labeled cells. SP-B declined to 50% of control in lavage at 12 h, but no changes in total lung SP-A and -B were seen. The number of SP-A positively labeled cells did not change, but SP-A label intensity measured by IHC in type II cells showed parallel results to Northern blots and ISH. The response of SP-A in Clara cells was similar. SP-B immunolabeling intensity rose in both type II and Clara cells throughout the exposure. SP-C ISH intensity fell at 12 h and was increased to two times control by 60 h of hyperoxia. Sharp declines in SP expression occurred by 12 h of 95% O2 and may affect local alveolar stability.