A vaccinia-specific target antigen for recognition of anti-vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus-cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first-phase and the second-phase target antigens were cross-reactive in cytotoxicity inhibition assays. Cowpox virus, CPR Cl strain, brought about a lower response than vaccinia virus, IHD-J strain, in both sensitization of CTL and formation of CTL-susceptible cells at both the first and the second phase. The cross-reactive, but inefficient, recognition of anti-vaccinia CTL for cowpox-infected cells suggested a slight difference in the target antigens of the two viruses. Attempts to identify the target antigen were then made by comparing the polypeptide composition of vaccinia virus, cowpox virus, and their recombinants. SDS-PAGE analysis of trypsin-activated viruses revealed 44K (cowpox)/45K (vaccinia) polypeptides which corresponded to the difference in target cell formation. Trypsinization of the viruses also increased the ability of the virus to induce the production of CTL-susceptible target cells.