Human cytomegalovirus (CMV) is a ubiquitous pathogen which sets up a lifelong persistent infection and which can lead to significant disease in the immunosuppressed. The immunological mechanisms controlling CMV in the long term are not defined completely, but CD8+ T lymphocytes are thought to play an important role. Antiviral CD8+ T lymphocytes may exist in very large pools in healthy individuals. Although the detailed composition of these pools is not completely understood, there is known to be heterogeneity, in particular of CD45 isoform expression. We have therefore investigated the CD8+ T-lymphocyte response against CMV directly ex vivo using Class I tetramers combined with stains for a range of phenotypic markers followed by four-colour flow cytometric analysis. In particular, we examined expression of these phenotypic markers in relation to the expression of CD45 isoforms. We found that a spectrum of phenotypes exists stably, from CD45R0(high)/RA(low) through CD45RA(high)/R0(low), and that expression of other surface markers such as CD28 and CD62L, and also TCR usage, may vary in parallel with CD45 isoform expression. In some individuals, expansions of antigen-specific CD8+ T lymphocytes bearing specific TCR Vbeta chains were restricted to cells of particular CD45 isoforms. Immunity against CMV comprises a large population of CD8+ T lymphocytes with heterogeneous potential, a spectrum in which CD45 isoform expression may play a central role.