Costimulatory interactions are important regulators of T-cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small-molecule inhibitors of the CD40–CD154 costimulatory protein–protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration-dependently inhibit the CD154(CD40L)-induced cellular responses in human THP-1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40–CD154 interaction with sub-micromolar activity, but it also has considerably more than 100-fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF-R1–TNF-α, BAFF-R(CD268)–BAFF(CD257/BLys), OX40(CD134)–OX40L(CD252), RANK(CD265)–RANKL(CD254/TRANCE), or 4-1BB(CD137)–4-1BBL. There is now sufficient structure-activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction.