In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF MS in combination with chemical and enzymatic treatments and MS/MS to analyze the neutral and anionic N-glycans of the mutant as compared to the wild type. The major neutral species were, as expected, of the composition Hex(10-11)HexNAc(2-3) with one or two terminal glucose residues. Consistent with the block in processing of neutral N-glycans caused by the absence of glucosidase II, fucose was apparently absent from the N-glycans and bisecting N-acetylglucosamine was rare. The major anionic oligosaccharides were sulfated and/or methylphosphorylated forms of Hex(8-11)HexNAc(2-3), many of which surprisingly lacked glucose residues entirely. As anionic N-glycans are considered to be mostly associated with lysosomal enzymes in Dictyostelium, we hypothesise that glycosidases present in the acidic compartments may act on the oligosaccharides attached to such slime mould proteins. Furthermore, our chosen analytical approach enabled us, via observation of diagnostic negative-mode MS/MS fragments, to determine the fine structure of the methylphosphorylated and sulfated N-glycans of the M31 glucosidase mutant in their native state.