In response to viral infection, unprimed naive CD8⁺, major histocompatibility complex class I—restricted, virus-specific T cells clonally expand and differentiate into memory- and effector-type cells. Changes in CD8⁺ subset distribution were studied in 17 subjects with acute human immunodeficiency virus type 1 infection and in 14 subjects with acute Epstein-Barr virus (EBV) infection, with combined CD45RO, CD27, and CD28 monoclonal antibodies. A vast expansion of memory-type CD45RO⁺CD27⁺CD8⁺ T cells, with high expression of the cell-cycle marker Ki-67, was observed in both infections. Strikingly, CD45RO⁺CD27⁺CD28⁻ cells increased >10-fold in acute viral infection and had high Ki-67 expression. In acute EBV infection, a substantial portion of the expanded T cells were EBV-peptide specific. These cells resided mainly in the CD45RO⁺CD27⁺ subpopulation, with most in the CD27⁺CD28⁻ subpopulation. Content of perforin expression, as a measure of cytotoxic capacity, was relatively low in the CD27⁺CD28⁺ T cells and highest in the CD27⁻CD28⁻ subpopulation.