CD69 is a disulfide‐linked homo‐dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross‐linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4⁺ T cell clone. Transfection of the cDNA clone in CV‐1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7‐kb mRNA in phorbol 12‐myristate 13‐acetate‐ or phytohemoagglutinin‐stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti‐mouse CD69 antibodies by secreting interleukin‐2 and interferon‐γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR‐P1 and Ly‐49 families of natural killer cell activation molecules.