This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40 % of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7 %, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for ’brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core α1−6-linked and outer-arm α1−3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewisx and NeuAcα2−3 substituted Galβ1−4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal β1−3-linked galactose residues was observed. The presence of β1−3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAcα2−3Galβ1−3(NeuAcα2−6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one β1−3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing β1−3-linked galactose and ’polylactosamine' N-glycans with one to three terminal β1−3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal β1−3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.