Activation of T cells is dependent upon coordinate engagement of Ag and costimulator receptors on their surfaces. In the case of the Ag receptors (TCRs), activation thresholds have been defined, with the number of TCRs that must be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have determined whether comparable activation thresholds exist for the costimulator receptors on T cells. To facilitate this type of quantitative costimulator analysis, we developed a novel two-step protein transfer approach that permits delivery of graded amounts of proteins to APC surfaces. By adding a human B7-1. Fcgamma1 (Fc domain of human IgG1) fusion protein to cells precoated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1. Fcgamma1 reincorporated into cell membranes by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surface density of B7-1. Fcgamma1. Significantly, the threshold B7-1. Fcgamma1 density required for cytokine production differed between IFN-gamma and IL-2 and mirrored the hierarchy (IFN-gamma < IL-2) described previously for the TCR activation threshold. Hence, this study invokes a novel protein transfer strategy to establish that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T cell responses. The notion of costimulator receptor activation thresholds emerges.