Incorporation of [3H]glucosamine and 35S into glycosaminoglycan (GAG) was measured in hamster lung explant cultures at 0, 1, 4, and 24 h after a single endotracheal instillation of Escherichia coli endotoxin. Lung content of GAG was measured in a second group of treated animals over an 8-day period. Albumin was detected after endotoxin treatment in bronchoalveolar lavage fluid at 24 h but was not found in lavage fluid 7 days later or in lavage fluid of saline-treated animals. Over the initial 24 h, increasing amounts of radiolabeled precursor molecules were incorporated into all classes of GAG. Proportionally more radiolabel was incorporated into hyaluronic acid and chondroitin sulfate, and less was incorporated into heparan sulfate. The proportion of radiolabel incorporated into dermatan sulfate did not change. Total lung content of hyaluronate and chondroitin sulfate was elevated at 24 h but was returning to baseline by 8 days. The lung content of dermatan sulfate was increased at 8 days; lung content of heparan sulfate did not change over the 8-day study period. Elevations in the amount of explant heparan sulfate that bound to antithrombin III (AT III) were found at 1 h after both saline and endotoxin treatment. Radiosulfated heparan sulfates were found in blood from hamsters treated with endotoxin 1 h previously; these heparan sulfates did not bind to AT III. However, blood contained heparin-like activity. We conclude that endotoxin differentially alters the metabolism of each class of hamster lung glycosaminoglycans and that metabolic changes begin very rapidly after endotoxin exposure. The relation of pulmonary endothelial injury to the presence of heparin-like activity in blood is not yet clear.