Using a biochemical technique, the authors characterized and identified the plasminogen activator (PA) derived from tissue extracts of six aural cholesteatomas. The results of fibrin zymography indicated that the tissue extracts of two cholesteatomas demonstrated two lytic zones on fibrin-agarose plates. One of the lytic zones was at about 72 kd, while the other zone was at about 64 kd. Using various goat immunoglobulin G (IgG)-containing antibodies (anti-human uterine tissue type PA (t-PA), anti-human low-molecular-weight (LMW) urokinase, and nonspecific goat IgG) and plasminogen-free fibrin-agarose plates, we confirmed that the cholesteatoma tissue extracts contained 72 kd t-PA and 64 kd urokinase type PA (u-PA). Furthermore, we measured the t-PA and u-PA activities in the tissue extracts selectively by parabolic rate assay. In order to estimate the PA activity, we developed optimal conditions for this assay. The specific t-PA activity ranged from 0.03 to 0.43 mIU/micrograms-protein and the specific u-PA activity ranged from 0 to 0.35 mIU/microgram-protein. The highest percentage of u-PA with respect to the total PA activity was 44.9%. However, in four of the six cases, we failed to detect u-PA activity. In the present study, we thus clarified the presence of PAs in tissue extracts of aural cholesteatomas. Furthermore, we confirmed that measureable u-PA occurred in some tissue extracts. We anticipate that the u-PA in inflammatory tissues plays an important role in the degradation of the extracellular matrix via the formation of plasmin and collagenases.