A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild type strain (X22) and a fucosylation-defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(α1–3)GlcNAc-α-1-P-Ser, was identified by electrospray ionization mass spectrometry and NMR. It was shown using monosaccharide and gas chromatography mass spectrometry analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose-deficient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identifyO-linked glycans (Champion, A., Griffiths, K., Gooley, A. A., Gonzalez, B. Y., Gritzali, M., West, C. M., and Williams, K. L. (1995) Microbiology 141, 785–797). Quantitative studies on wild type SP96 indicated that there were approximately 60 sites with phosphodiester-linkedN-acetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues were not found to be modified.