Activation of primary resting T cells requires costimulation which can be delivered by the B7 molecules (CD80 and CD86) expressed on activated antigen-presenting cells (APC). In the present study, we examined in vitro effects of immunotoxins (ITs) composed of gelonin conjugated to mAbs against CD80 or CD86 (alphaCD80-IT and alphaCD86-IT). The specificity of both ITs was demonstrated using CD80 and CD86 transfected cell lines. In primary mixed lymphocyte cultures (MLCs), it was found that the average inhibitory capacity of alphaCD86-IT (72%) and alphaCD80-IT (30%) was significantly higher than alphaCD86 (54%) and alphaCD80 (11%). In reculture MLC experiments it was found that peripheral blood mononuclear cells pretreated with alphaCD86/alphaCD80 regained full stimulatory capacity whereas alphaCD86-IT/alphaCD80-IT pretreatment induced >95% loss of stimulatory capacity. Our results therefore demonstrate that these alphaB7-ITs functionally block B7-CD28 costimulatory signaling and eliminate activated APC.