In order to define the function of the CD 5 (T1, Leu 1, Tp 67 in the human; Ly‐1 in the mouse) molecule, a cDNA clone of human CD 5 was expressed in a CD 5‐deficient Jurkat cell line and in a murine T cell hybridoma. A Jurkat subclone (Jurkat 9.9) produced interleukin 2 (IL 2) in response to anti‐CD 3 monoclonal antibody (mAb) cross‐linked to solid support. IL 2 production was enhanced by co‐culture with the anti‐CD 5 mAb OKT1. A CD 5‐deficient mutant clone Jurkat 1.15 was generated by treatment with ethyl methanesulfonate followed by selection with anti‐CD 5 mAb plus complement. Jurkat 1.15 did not demonstrate enhancement of IL 2 production by OKT1 in the presence of cross‐linked anti‐CD 3 mAb. A cDNA encoding human CD 5 was introduced into a defective retrovirus which was used to infect Jurkat 1.15. A Jurkat clone stably expressing CD 5 was established. In response to OKT1, a rise in intracellular calcium was observed in both the parent Jurkat 9.9 and the CD5− infectant but not in the CD5‐ mutant or a G418‐ resistant control. Furthermore, expression of CD 5 restored the augmentation of IL 2 production by OKT1 in response to cross‐linked anti‐CD 3 mAb. A murine T cell hybridoma By 155.16 which produces IL 2 in response to HLA‐DR antigens was also infected with the CD 5‐recombinant retrovirus and three stable CD5− infectants were generated. These hybridomas showed enhancement of IL 2 production by stimulation with OKT l in the presence of suboptimal concentrations of soluble anti‐murine CD 3 mAb. These results provide further evidence that CD 5 provides a co‐stimulatory signal for T cell activation.