Polyclonal stimulation of CD8+ cytotoxic T lymphocytes (CTL) occurs during infection with many viruses including those not known to transform CTL or encode superantigens. This polyclonal CTL response includes the generation of high levels of allospecific CTL directed against many class I haplotypes. In this report we investigated whether the allospecific CTL generated during an acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6 mice were stimulated specifically by antigen recognition or nonspecifically by polyclonal mechanisms possibly involving lymphokines or superantigens. An examination of the ability of different strains of mice to induce high levels of CTL specific for a given alloantigen showed that most, but not all, strains generated high levels of allospecific CTL, and that their abilities to generate them mapped genetically to the major histocompatibility complex locus, exclusive of the class II region. This indicated that the virus-induced allospecific CTL generation was independent of the class II allotype, and mice depleted of CD4+ cells generated allospecific CTL, indicating independence of class II-CD4+ cell interactions and resulting CD4+ cell-secreted lymphokines. FACS staining with a variety of V beta-binding antibodies did not show a superantigen-like depletion or enrichment of any tested V beta + subset during infection. Several experiments provided evidence in support of direct stimulation of CD8+ cells via the T cell receptor: (a) both virus- and allo-specific killing were enriched within a given V beta subpopulation; (b) relative CTL precursor frequencies against different class I alloantigens changed during the course of virus infection; (c) the relative levels of virus-induced, allospecific CTL-mediated lysis at day 8 after infection did not parallel the CTL precursor frequencies before infection; and (d) limiting dilution analyses of day 8 LCMV-infected spleen cells stimulated by virus-infected syngeneic peritoneal exudate cells (PEC) revealed not only the expected virus-specific CTL clones, but also a high frequency of clones that were cross-reactive with allogeneic and virus-infected syngeneic targets. In addition to the virus cross-reactive allospecific CTL clones, virus-infected PEC also stimulated the generation of some allospecific clones that did not lyse virus-infected fibroblasts. Surprisingly, LCMV-infected PEC were much more efficient at stimulating allospecific CTL clones from day 8 LCMV-infected splenocytes than were allogeneic stimulators. These results indicate that at least part of the polyclonal allospecific CTL response elicited by acute virus infection is a consequence of the selective expansion of many clones of allospecific CTL which cross-react with virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)