Exposure of adult rats to 100% O2 produces a lethal injury by 72 h. We reasoned that matrix metalloproteinases participate in the pathogenesis of hyperoxic lung injury. To that end we studied the expression and activity of gelatinases A and B and interstitial collagenase in lung tissues and bronchoalveolar lavage fluids (BALF) of rats exposed to 100% oxygen for 60 h. Gelatin zymography of BALF samples revealed a 472 kDa molecular species both in controls and oxygen-exposed animals. In addition, BALF from hyperoxic rats exhibited a 95-kDa gelatinase. Likewise, BALF total gelatinolytic and collagenolytic activities were significantly increased in oxygen-exposed rats. In situ hybridization revealed an increase in type IV collagenases as well as interstitial collagenase mRNAs in the oxygen-exposed lungs. The three enzymes were expressed by alveolar macrophages, and in variable degrees by interstitial and alveolar epithelial cells. Immunoreactive gelatinase B and collagenase paralleled the cell localization of the mRNAs but were also detected in the alveolar walls and interstitium. In situ zymography showed gelatinolytic activity in frozen sections of oxygen-exposed lungs but not in normal lungs. The upregulation of these metalloproteinases during acute exposure to 100% O2 suggests that they might contribute to hyperoxic lung damage through the degradation of extracellular matrix components.