Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by ¹H and ¹³C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as β-glucopyranosylceramides (GlcCer) having (4E,8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2‘-hydroxyoctadecanoate or N-2‘-hydroxy-(E)-3‘-octadecenoate. The mycelium form GlcCer had both fatty acids in a ∼1:1 ratio, while that of the yeast form had on average only ∼15% of the (E)-Δ³-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have ∼70% and ∼90% N-2‘-hydroxy-(E)-3‘-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2‘-hydroxy-(E)-3‘-octadecenoate at only ∼20 and ∼50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764−8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.