Although a number of studies on the phenotypic changes that occur after T-cell activation have already been published, the specific immunophenotypic features of T-lymphocytes and the frequency at which TCR-variable region (TCR-V) restricted T-cell expansions occur “in vivo” during acute viral infection still remains to be established. We report on the immunophenotype and TCR-V repertoire of peripheral blood T-cells from 28 patients with acute infectious mononucleosis. Immunophenotypic studies were performed by flow cytometry using direct immunofluorescence techniques and stain-and-then-lyse sample preparation protocols with three- and four-colour combinations of monoclonal antibodies directed against a large panel of T- and NK-cell associated markers, activation- and adhesion-related molecules and TCR-Vβ, -Vγ and -Vδ families. Nearly all patients (27/28) showed a massive expansion of CD8 +/TCRαβ + T cells, the majority (>90%) of which displayed an immunophenotype compatible with T-cell activation: CD2 +high, CD7 +low, CD11a +high, CD38 +high, HLA-DR +high, CD28 +/−low, CD45RO +high, CD45RA −/+low, CD11b −/+low, CD11c +/−low, CD16 −, CD56 −, CD57 −, CD62L −, CD94 −, CD158a −, CD161 −, NKB1 −. Additionally, the levels of both CD3 and CD5 were slightly decreased compared to those found in normal individuals. Late-activation antigens, such as CD57, were found in small proportions of CD8 +/TCRαβ + T-cells. Increased numbers of CD4 +/TCRαβ + T-cells, TCRγδ + T-cells and NK-cells were also noticed in 17, 16 and 13 of the 28 cases studied, respectively. Evidence for activation of CD4 +/TCRαβ + and TCRγδ + T-cells relied on changes similar to those described for CD8 +/TCRαβ + although less pronounced, except for higher levels of both CD5 and CD28 in the absence of reactivity for CD11c on CD4 +/TCRαβ + T-cells and higher levels of CD161 and CD94 on TCRγδ + T-cells. Small expansions of one or more TCR-Vβ families accounting for 12 ± 7% of either the CD8 +/TCRαβ + or the CD4 +/TCRαβ + T-cell compartment were found in 12 of 14 patients studied, whereas the distribution of the TCR-Vγ and -Vδ repertoires tested in 2 of the individuals with expanded TCRγδ + T-cells was similar to that observed in control individuals. The results presented here provide evidence for an extensive T-cell activation during acute viral infection and establish the immunophenotype patterns associated with this condition.