Previous studies have determined that proteases are important in cold preservation injury to the liver. The purpose of this study was to determine the role of matrix metalloproteinases (MMPs) in cold preservation injury. Effluents were collected from rat livers after various periods of preservation either in Eurocollins solution or in University of Wisconsin (UW) solution. Effluents were also collected from 17 human donor livers stored in UW solution. To determine whether sinusoidal endothelial cells released MMPs when placed in the cold, these cells were isolated from rat livers and cultured at 4°C. Gelatin zymography, quantitative assay of gelatinolytic activity, immunoprecipitation, and Western blotting were used to identify metalloproteinases and to measure their activity. Human and rat liver effluents contained gelatin-digesting bands on zymography. Their appearance was inhibited by specific metalloproteinase inhibitors and also by lactobionate, the major ingredient of UW solution. The most prominent bands in humans and the rat appeared at approximately 72 kd and 92 kd, suggesting that they were the MMPs 72-kd gelatinase and 92-kd gelatinase. Supernatants of isolated rat sinusoidal endothelial cells stored in the cold contained similar bands. In the rat, the proteinases were present in both latent and active forms, but, in humans, predominately the latent form was seen. In humans, there were four prominent bands in the gelatin zymography. By immunoprecipitation, two of the bands were identified as the 92-kd gelatinase and a dimer or polymer of 92-kd gelatinase. Using Western blotting with a monoclonal antibody, a third band was identified as 72-kd gelatinase. In quantitative terms, gelatinolytic activity increased with time of cold storage in humans and in the rat. In the rat, gelatinolytic activity was greater when Eurocollins was the preservative than when UW solution was used. Taken together, these results indicate an important role for MMPs in the injury produced by cold preservation of the liver.