Dendritic cell (DC)-based immunotherapeutics must induce robust CTL capable of killing tumor or virally infected cells in vivo. In this study, we show that RNA electroporated post maturation and coelectroporated with CD40L mRNA (post maturation electroporation (PME)-CD40L DC) generate high-avidity CTL in vitro that lyse naturally processed and presented tumor Ag. Unlike cytokine mixture-matured DC which induce predominantly nonproliferative effector memory CD45RA(+) CTL, PME-CD40L DC prime a novel subset of Ag-specific CTL that can be expanded to large numbers upon sequential DC stimulation in vitro. We have defined these cells as rapidly expanding high-avidity (REHA) CTL based on: 1) the maintenance of CD28 expression, 2) production of high levels of IFN-gamma and IL-2 in response to Ag, and 3) the demonstration of high-avidity TCR that exhibit strong cytolytic activity toward limiting amounts of native Ag. We demonstrate that induction of REHA CTL is dependent at least in part on the production of IL-12. Interestingly, neutralization of IL-12 did not effect cytolytic activity of REHA CTL when Ag is not limiting, but did result in lower TCR avidity of Ag-reactive CTL. These results suggest that PME-CD40L DC are uniquely capable of delivering the complex array of signals needed to generate stable CD28(+) REHA CTL, which if generated in vivo may have significant clinical benefit for the treatment of infectious disease and cancer.