Because certain proteolytic enzymes are thought to be released by malignant cells, we have measured the activity of cathepsin B in the urine samples of 57 patients with gynecologic malignancies and 60 disease-free controls. A unit (U) of enzyme activity is the release of one n-mol of 7-amino-4-fluoromethylcoumarin (AFC) from BZ-val-lys-lys-arg-MNA min-1 ml-1. Units of activity in the malignant group (10.6 +/- 9.8) differed significantly (p less than 0.0001) from controls (2.8 +/- 3.3). Although enzyme activity in both groups correlated with increasing age, the difference between those subjects with malignancies and those with none remained significant (p = 0.049) by analysis of covariance after adjusting for age. There was no correlation between titers and the race or weight of the subjects in either group. Enzyme activity of subjects with malignant disease correlated (p = 0.003) with the clinical stage of disease. Optimum sensitivity and specificity as determined by Receiver Operator Characteristic Analysis with an upper normal level of 5 U were 84.2% and 86.7%, respectively. Our findings suggest that measurement of urinary cathepsin B might be useful in detecting and managing patients with gynecologic tumors.