We have analyzed cytotoxic thymus-derived lymphocyte (CTL) responses to vesicular stomatitis virus (VSV) to determine whether VSV precursor CTL (pCTL) can be primed in vivo in the absence of CD4+ cells. Our studies demonstrated that secondary anti-VSV CTL responses in vitro were markedly reduced by CD4-depletion prior to priming in vivo with VSV. Limiting dilution analysis indicated that the vast majority (> 90%) of VSV pCTL failed to become primed when exposed to VSV in the absence of CD4+ cells. A second minor population (5-10%) of pCTL was identified that was reproducibly primed in CD4-deficient mice. In contrast to CD4-depleted mice infected with free, infectious virus, CD4-deficient mice primed with VSV-infected, activated B cells mounted normal secondary anti-VSV CTL responses in vitro. Precursor estimates indicated that virtually all VSV pCTL became primed using this cellular immunogen. CD4-independent priming could not be achieved using VSV-infected, activated T cells, another permissive cell type for VSV replication. Thus, most VSV pCTL require inductive signals from classical CD4+ helper T cells in order to become primed in vivo and this requirement may be regulated in vivo by the antigen presenting cell.