CD3 antibodies are proven immunosuppressants capable of reversing transplant rejection episodes. Their general application has been limited both by their immunogenicity and, in particular, by the “first-dose” cytokine-release syndrome experienced by patients after the initial administration of antibody. We have produced a set of variants of the humanized YTH 12.5 CD3 monoclonal antibody (mAb)(Routledge et al., Eur. J. Immunol. 1991. 21: 2717) bearing different human heavy (H) chain constant regions, with the intention of finding a form of the antibody that is not able to activate T cells. Comparison of the variants having γl, γ2, γ3 and γ4 H chains in a competitive binding assay showed that antibody avidity was not affected by IgG subclass. Using a sensitive indicator of FcR binding activity (the capacity of the CD3 mAb to redirect cytotoxic T cells to kill the monocytic cell line U-937) we demonstrated a functional hierarchy of γl= γ4 α2= γ3 mb> γ2. An aglycosyl version of the γl CD3 mAb, produced by site-directed mutagenesis (Asn 297 to Ala), still had considerable activity in this assay (intermediate to the γl and α2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental γl form. When we tested their ablity to stimulate T cell proliferation in vitro in the presence of 5% human serum, all of the wild-type immunoglobulin isotypes were found to be active, although there were T cell donor-dependent variations in the extent of the responses. The aglycosyl γl mAb was, however, completely non-mitogenic in all of ten donors tested, unless the assay was performed in IgG-free medium. Despite being non-stimulatory, this mAb was also able to inhibit the mixed lymphocyte reaction responses of both naive and primed T cells. Comparison of the yl and aglycosyl γ1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the γl constant region reduced the in vivo tumor necrosis factor-a response by a factor of at least 16-fold. These data suggest that the aglycosyl γl CD3 mAb is a promising candidate for immunosuppressive therapy without “first dose” side effects.