Prostate-specific antigen (PSA) is a chymotrypsin-like serine protease, which dissolves the coagulum forming after ejaculation. In seminal plasma, PSA occurs as a free enzyme comprising several isoenzymes. Two glycosylation variants of intact PSA (A and B) and three nicked forms (C, D and E) can be separated by anion exchange chromatography. About 70% of free PSA is enzymatically active and about 30% is inactive as a result of nicking. 60-95% of the immunoreactive PSA in serum occurs in complex with α1-antichymotrypsin (ACT). A few percent occurs in complex with α1-proteinase inhibitor and the rest is free. In serum from patients with prostate cancer the proportion of complexed PSA is higher and that of free PSA lower than in benign prostatic hyperplasia (BPH). By measuring the proportion of complexed to total or free to total PSA, 30-50% of the false positive results due to BPH can be eliminated. The reason for the higher proportion of complexed PSA in prostate cancer is not known. We present the hypothesis that a higher proportion of PSA released from hyperplastic tissue than from cancer tissue is nicked and less able to complex with ACT. Complexes of PSA with α2-macroglobulin (A2M) have been detected by immunoblotting but these are not detected by presently available immunoassays. The significance and clinical utility of PSA-A2M complexes is not known, but because protease-A2M complexes are removed from the circulation very rapidly, the concentrations of them may be very low in vivo. In vitro the reaction between PSA and A2M is rapid. If the sample contains significant amounts of active PSA, the complex formation may continue after sampling, causing loss of PSA immunoreactivity and change in the proportion of free and complexed PSA. The effect of this and other preanalytical factors affecting the PSA concentrations in serum needs to be considered when using PSA for early diagnosis of prostate cancer.