Human prostate-specific antigen (hPSA) is found in serum and semen in a variety of forms, is form-free and complex, and has clinical importance to prostate cancer diagnosis. Here, a simple procedure is described for the efficient purification of hPSA from seminal plasma. The semen was clarified by centrifugation, and the isolation of PSA was carried out by immunoaffinity chromatography using the monoclonal antibody anti total PSA CB-PSA.4. The recuperation of PSA from seminal plasma by this procedure was 66.4%, and a purification factor of 65.8 was reached. The specificity activity obtained was 0.79 mg PSA/mg protein, and the preparation appeared homogeneous by SDS-PAGE and immunoelectrophoresis. A molecular weight of preparation was 30.46 kDa by SDS-PAGE, similar to the commercial PSA. These results indicate that immunoaffinity purification of PSA by means of this monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified human PSA.