The induction of toll-like receptor 7 (TLR7)-dependent type I interferons (IFN-α/β) from plasmacytoid dendritic cells as well as the production of TLR8-dependent type II interferon (IFN-γ), TNF-α, and IL-12 in myeloid dendritic cells are of importance in generating T helper-1 biased adaptive immune responses. In an effort to identify novel dual TLR7/TLR8-active compounds, we undertook structure-activity relationship studies in pyrimidine 2,4-diamines, focusing on substituents at C5. Several analogues substituted with aminopropyl appendages at C5 displayed dominant TLR8-agonistic activity. N4-Butyl-6-methyl-5-(3-morpholinopropyl)pyrimidine-2,4-diamine was found to be a very potent dual TLR7/TLR8 agonist. Employing novel cytokine reporter cell assays, we verified that potency at TLR7 correlates with IFN-α/β production in human blood, whereas IFN-γ and TNF-α induction is largely TLR8-dependent. Dual TLR7/TLR8 agonists markedly upregulate CD80 expression in multiple dendritic cell subsets, providing insight into the immunological basis for the superior adjuvantic properties of such innate immune stimuli.