CD40 Ligand (CD40L) is transiently expressed on the surface of T‐cells and binds to CD40, which is expressed on the surface of B‐cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B‐cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T‐cell dependent B‐cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X‐ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X‐ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, El14, El17). These residues were studied experimentally through site‐directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, El 14R, El 17R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, El 17N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge‐reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74‐R203, D84‐R207, and El 17‐R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.