Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.