We have examined the extent of allelic exclusion at the T cell receptor (TCR)β locus using monoclonal antibodies specific for Vβ products. A small proportion (-1%) of human peripheral blood T cells express two Vβ as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding Vβ chains. Dual β T cells are present in both the CD45R0+ and CD45R0− subset. These results indicate that dual β expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCRβ allelic exclusion is dependent only on asynchronous rearrangements at the β locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional β protein.