The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c high DC subsets. CD4+, CD8α+, and CD8α− CD4−(double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4+ and CD8α+ DC subsets showed distinct basal expression profiles differing by> 200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4+ DC, differing from them by< 25 genes. This suggests that CD4+ and DN DC are closely related phylogenetically, whereas CD8α+ DC represent a more distant lineage, supporting the historical distinction between CD8α+ and CD8α− DC. However, staining patterns revealed that in contrast to CD4+ DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c high DC subsets that to date have been difficult to define as rigid distinctions.