We studied a simple heat treatment method for measuring free prostate-specific antigen (PSA). Samples were incubated at 56, 58, and 60 degrees C for 5, 15, 30, 45, and 60 min. Then, 1 ml samples were fractionated on a Sephacryl S-200 gel filtration column to separate alpha1-antichymotrypsin-complexed PSA (ACT-PSA) and free PSA. Values of ACT-PSA decreased with increasing incubation temperature and time, whereas free-PSA remained relatively constant. The optimal temperature and time for incubation were 58 degrees C and 30 min. Using free/total-PSA ratios, we were able to distinguish between benign prostatic hyperplasia and prostate carcinoma in patients whose PSA was in the diagnostic 'grey zone', i.e. 4.1 to 10.0 ng/ml. Through receiver operating characteristic curve analysis, the area under the curve increased from 0.675 to 0.871 when comparing the performance of total PSA to the free/total-PSA ratio. Thus, clinical application of our present methodology may reduce the need to obtain prostatic biopsies in patients whose PSA level is within the diagnostic 'grey zone'.