During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4+CD25+). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vbeta usage. The expression of Foxp3 and CD127 in CD4+CD25(high) T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vbeta usage in both study and control groups. Significant variability was found in Vbeta usage for CD4+ and CD8+ T cells when the distribution of the percentage of T cells expressing each Vbeta family was analysed between individuals within each group (P < 0.001; Kruskal-Wallis). Significant difference was also found in average usage of Vbeta2, Vbeta5.1 and Vbeta14 chains within CD4+ T cells and Vbeta2, Vbeta8 and Vbeta21.3 chains within CD8+ cells between the groups (P < 0.05; Student's t-test). There was no difference between the two groups with regard to the proportion of CD4+CD25(high) T cells and no difference in the average expression of Foxp3 or CD127 within the CD4+CD25(high) population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vbeta usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.