Despite the widely accepted use of prostate specific antigen (PSA) as a marker of prostate cancer, this molecule has not yet been completely characterized. Past studies have well established, however, using both amino acid and cDNA sequencing techniques, that PSA contains 237 amino acids, with a molecular mass of 26,079 Da for the peptide moiety of the molecule. The present study reports analysis of this protein by ion spray mass spectrometry (ISMS) and analysis of its carbohydrate moiety by NMR spectroscopy. The predominant PSA molecular species detected by ISMS was at relative molecular mass (M(r)) of 28,430, indicating that PSA contains a carbohydrate residue of M(r) 2,351, for a total percentage of carbohydrate of 8.3%. Analysis of PSA by SDS-PAGE, however, showed a M(r) of 32,000 to 33,000, suggesting an overestimation of the molecular weight by the latter technique. The complete primary structure of the PSA carbohydrate chain was determined by NMR spectroscopy in combination with carbohydrate composition analysis. The experimentally determined carbohydrate content of PSA confirms that only one N-glycosylation site is occupied in the protein. The proposed carbohydrate structure is a diantennary N-linked oligosaccharide of the N-acetyllactosamine type, with a sialic acid group at the end of each of the two branches. In addition, our data indicate that approximately 70% of the PSA molecules contain a fucose group in the core chitobiose moiety. The calculated molecular weight of this carbohydrate structure (M(r) 2,351.8) is in excellent agreement with the predicted molecular weight of the carbohydrate group, based on the M(r) 28,430 for PSA measured by ion spray mass spectrometry and M(r) 26,079 calculated from the consensus sequence for the peptide portion of the molecule. ISMS of PSA is thus proposed as a convenient and reliable method of quality control, an indispensible step towards international standardization of this very important tumor marker for detection and monitoring of prostatic diseases, especially prostate cancer.