The presence of plasminogen activator (PA) inhibitor in human articular cartilage extracts was shown using a microtiter plate assay using immunofixed urokinase. Cartilage urokinase inhibitor had a molecular weight of 66,000 on gel chromatography. Cartilage extracts also contained alpha 1-proteinase inhibitor; however, the urokinase inhibitor was distinguishable from such serum inhibitors immunologically. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin overlay, inhibition of urokinase was observed accompanying higher molecular weight complex formation. The cartilage urokinase inhibitor was unstable with acid, heat and SDS treatment, and required the active site of urokinase for inhibition.