We have applied MHC class I tetramers representing the two H2 b MHC class I-restricted epitopes of the mouse male-specific minor transplantation Ag, HY, to directly determine the extent of expansion and immunodominance within the CD8+ T cell compartment following exposure to male tissue. Immunization with male bone marrow (BM), spleen, dendritic cells (DCs) and by skin graft led to rapid expansion of both specificities occupying up to> 20% of the CD8+ T cell pool. At a high dose, whole BM or spleen were found to be more effective at stimulating the response than BM-derived DCs. In vivo, immunodominance within the responding cell population was only observed following chronic Ag stimulation, whereas epitope immunodominance was established rapidly following in vitro restimulation. Peptide affinity for the restricting MHC molecule was greater for the immunodominant epitope, suggesting that this might be a factor in the emergence of immunodominance. Using tetramers, we were able to directly visualize the cross-primed CD8+ HY response, but we did not find it to be the principal route for MHC class I presentation. Immunization with female spleen or DCs coated with the full complement of defined HY peptides, including the A b-restricted CD4+ Th cell determinant, failed to induce tetramer-reactive cells.