The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vbeta gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vbeta families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4(+) and CD8(+) T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8(+) CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4(+) or CD8(+) T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vbeta families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.