Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostatespecific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA-and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 g/L. The median total hK2 concentration was 0.022 g/L (range, 0.0015-0.37 g/L). hK2 concentrations were 0.1-58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41-50 and 51-60 years of age. The ratio of hK2 to PSA steadily decreased from 5-30% at PSA <1 g/L to 1-2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 g/L (range, 0.0015-16.2 g/L). The median free hK2 concentration was 0.070 (range, 0.005-12.2) g/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%).