To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40‐Fc fusion protein was able to specifically bind to CD4⁺ and various CD8⁺ T cell clones activated with immobilized anti‐CD3. The ¹²⁵I‐labeled CD40‐Fc fusion protein bound anti‐CD3 activated CD4⁺ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10‐20 nM. The human CD40‐binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8⁺ blood mononuclear T cells activated by anti‐CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40‐Fc obtained after anti‐CD3 activation.