Quantitative immunoblotting of prostate cancer patient sera revealed that most prostate specific antigen was in complexes with alpha 1-antichymotrypsin or alpha 2-macroglobulin with little of it being free antigen. Complexes of prostate specific antigen with these protease inhibitors in patient sera comigrated during electrophoresis with the respective purified complexes. Each complex was selectively removed from patient sera by absorption with specific antibodies. When prostate specific antigen was added to normal plasma, complexes with alpha 2-macroglobulin appeared first and after 1 hr, the distribution was approximately 40% free antigen, approximately 40% complexes with alpha 2-macroglobulin, and approximately 20% complexes with alpha 1-antichymotrypsin. These data show that prostate specific antigen reacts more readily with alpha 2-macroglobulin than with any other protease inhibitor in plasma and that the antigen complexes with alpha 2-macroglobulin in vivo in cancer patients.