CD80 and CD86 both costimulate T cell activation. Their individual effects in vivo are difficult to study as they are coordinately up-regulated on APCs. We have studied mice expressing rat insulin promoter (RIP)-CD80 and RIP-CD86 on the NOD and NOD. scid genetic background to generate in vivo models, using diabetes as a readout for cytotoxic T cell activation. Accelerated spontaneous diabetes onset was observed in NOD-RIP-CD80 mice and the transfer of diabetes from 6-wk-old NOD mice to NOD. scid-RIP-CD80 mice was greater compared with NOD-RIP-CD86 and NOD. scid-RIP-CD86 mice, respectively. However, the secondary in vivo response was maintained if T cells were activated through CD86 costimulation compared with CD80. This was demonstrated by greater ability to cause recurrent diabetes in NOD-RIP-CD86 diabetic mice transplanted with 6-wk-old NOD islets and adoptively transferred diabetes from diabetic NOD-RIP-CD86 mice to NOD. scid mice. In vitro, CD80 costimulation enhanced cytotoxicity, proliferation, and cytokine secretion in activated CD8 T cells compared with CD86 costimulation. We demonstrated increased CTLA-4 and programmed death-1 inhibitory molecule expression following costimulation by both CD80 and CD86 (CD80> CD86). Furthermore, T cells stimulated by CD80 were more susceptible to inhibition by CD4+ CD25+ T cells. Overall, while CD86 does not stimulate an initial response as strongly as CD80, there is greater sustained activity that is seen even in the absence of continued costimulation. These functions have implications for the engineered use of costimulatory molecules in altering immune responses in a therapeutic setting.