We have studied the costimulation requirements for the generation of cytotoxic T (Tc) cells in an in vitro recall response to alloantigens. Firstly, we demonstrate that recombinant vaccinia viruses encoding class I MHC can stimulate primary in vivo responses and prime for secondary in vitro responses specific for the immunizing alloantigen. The secondary in vitro response comprises both naive and memory components that are distinguishable kinetically. Naive alloreactive Tc cell precursors are dependent upon the presence of CD80 on the in vitro stimulating population for activation and generation of effector function, as described previously. However, Tc cells from animals primed in vivo with vaccinia virus (VV) encoding allo-MHC do not require CD28-CD80 interactions to respond to the alloantigen presented in vitro. This finding provides further evidence that memory Tc cells have less stringent activation requirements in vitro than naive cells. From limiting dilution analysis of the relative contribution of naive and memory Tc cell precursors in 'primary' responses, to MHC class I alloantigen, memory alloreactive Tc cell precursors, possibly primed by cross-reactive environmental antigens, contribute approximately one-fifth of the precursors. Memory responses exhibit similar precursor frequencies as primary responses. Thus, we conclude that memory is largely a result of qualitative rather than quantitative changes in Tc cell precursors.