Inaccurate determination of total prostate-specific antigen (t-PSA) mainly derives from inadequate estimation of this heterogeneous molecule and its complexes with serum protease inhibitors, such as the a,-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M). While ACT-PSA complex is confirmed to be an important quantity for prostate cancer (PCa) diagnosis, alpha 2M-PSA complex is still an unmeasurable fraction. This study was performed to evaluate the phycicochemical conditions of PSA complex formation with serum protease inhibitors, focusing on ACT and alpha 2M. t-PSA and free prostate-specific antigen (f-PSA) levels were estimated with commercial immunoassays (Axsym/Abbott, Enzymun/Boehringer Mannheim, Elfa/ Biomerieux), while the formation of PSA complexes with ACT and alpha 2M with Western-blot electrophoresis. t-PSA values were unexpectedly lower after incubation of semen PSA for 4 days at 4 degrees C or at 37 degrees C in female serum and goat serum relevant to incubation in BSA buffer, possibly due to immunoreactivity loss in alpha 2M-PSA formation in the serum matrices. The transformation of PSA molecule into various measured and unmeasured forms after exposure to serum, especially suggested the inadequacy of serum matrices for f-PSA standard preparation. Loss of stability in PSA complexes was observed after dilution of prostate cancer (PCa) serum in aqueous buffer (BSA buffer), possibly due to dissociation of complex structure, the effect being mended by prior excess addition of ACT or alpha 2M. Optimum pH (approximately 7.5) and temperature (37 degrees C) for serum protease inhibitors binding to PSA were these of human serum, the complex formation increasing with incubation time, but not with PSA concentration.