We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).