The predominant acidic coagulum protein of freshly ejaculated human semen under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was purified for the first time from washed seminal coagulum keeping urea throughout the entire procedure. Washed seminal coagulum was isolated from fresh semen sample in acetate buffer at pH 4.5 at 0 degrees C, solubilized rapidly in deionized 4 M urea followed by reduction and carboxymethylation of disulfide bonds. By CM-Sephadex and Sephacryl S-300 high-resolution chromatography in presence of urea at room temperature, the predominant acidic coagulum protein was highly purified (99.1%) with a yield of 2.7% of the coagulum protein. The molecular weight of the purified protein in reducing SDS-PAGE was 162 kD.