We have previously isolated two extracellular cysteine proteases (60 and 30 kDa, respectively) from the cell filtrate of an isolate of Trichomonas vaginalis. In this study the clinical presentation of 12 clinical isolates of T. vaginalis was correlated with their protease activity. All 12 isolates produced a 60-kDa protease as demonstrated by immunoblotting. However, only 5 of 12 isolates produced a 30-kDa protease in the extracellular filtrate. Protease activity did not differentiate between isolates obtained from symptomatic versus asymptomatic women. The 60-kDa protease, which breaks down into a 43- and 23-kDa subunit, was detected by immunoblotting with anti-23-kDa cross-reacting rabbit serum in the vaginal washes of 3/3 women with active T. vaginalis infection, 0/1 woman cured of T. vaginalis and 0/2 control women with vaginal candidiasis. These results suggest that the 60-kDa protease is found in all isolates testes in vitro, is present in active disease and may be important in the pathogenesis of T. vaginalis infection.