The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expression of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate V beta independent of CD40L expression. Nevertheless, CD40L-mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L- T cells to SAG in vitro is also impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step in the life cycle of MMTV. The initial step in SAG-dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells.