The current assays for serum prostate specific antigen (PSA) have failed to produce the same PSA values on the same specimens because of problems with antibody specificity and calibrator preparation. To eliminate these problems, we proposed to replace the current serum PSA assay with an assay specific for the PSA-ACT (PSAal-antichymotrypsin) complex in the serum. An assay specific for the PSA-ACT complex was established using the anti-PSA antibody to coat the microplate for capturing the PSA complex and anti-ACT polyclonal antibodies for quantification. There was an excellent correlation between serum concentrations of PSA-ACT and total PSA, using either the Hybritech calibrator (y = 0.996) or a serum calibrator prepared in house (y = 0.993), in random as well as in serial specimens from 14 individual patients. Even though we did not find a gradual increase in the percentage of PSA-ACT with the increase of total PSA in cancer patients, a slightly higher percentage of free PSA was measured in pooled normal sera (18%) and in pooled sera containing only 12 ng/ml of total PSA (12%), compared to serum pools containing elevated PSA ( ~1 0 0 ng/ml) level, in which most PSA was in the complex form (95%). Therefore, using an assay that specifically measures the PSA-ACT complex in the serum not only simplifies the preparation of calibrator but eliminates the difficulty of antibody selection. it also allows various assay kits to produce identical PSA values and also improve the test specificity for prostate cancer. o 1995 Wiley-Liss, Inc.