The hydroxyl radical (.OH) has been implicated as a cause of lung injury following asbestos exposure. However, despite in vitro evidence associating asbestos with .OH production, there has been no demonstration of such generation in vivo. We tested the hypothesis that .OH is formed in the lungs of rats exposed to asbestos in vivo by using salicylate as a free radical trap. Asbestos was instilled intratracheally, and control rats were sham-exposed. Six to seven days after exposure, the rats were given salicylate, the lungs were isolated, and salicylate hydroxylation products (2,3- and 2,5-dihydroxybenzoic acid), reflecting .OH production, were measured. There was significantly more 2,3-dihydroxybenzoic acid in asbestos-exposed lungs compared with control lungs (2.32 +/- 0.360 nmol/lung versus 0.292 +/- 0.125, respectively, P < 0.001) and 2,5-dihydroxybenzoic acid (9.69 +/- 1.65 nmol/lung versus 2.63 +/- 0.274, respectively, P < 0.001). To demonstrate that the dihydroxybenzoic acid was actually formed in the lungs, the lungs from asbestos-exposed and control rats were isolated and perfused with either salicylate or 2,3-dihydroxybenzoic acid. In the lungs perfused with salicylate, 2,3- and 2,5-dihydroxybenzoic acids were detectable only in asbestos-exposed lungs. In the isolated lungs perfused with 2,3-dihydroxybenzoic acid, there was no significant difference in 2,3-dihydroxybenzoic acid between asbestos-exposed and control lungs. We conclude that asbestos stimulates .OH production in lungs in vivo.