Lack of allelic exclusion in the T-cell receptor (TCR) alpha locus gives rise to 2 different TCRs in 10% to 30% of all mature T cells, but the significance of such dual specificity remains controversial. Here we show that human CD4(+)CD25(+) regulatory T (Treg) cells express 2 distinct V alpha chains and thus 2 TCRs at least 3 times as often as other T cells. Extrapolating from flow cytometric analysis using V alpha 2-, V alpha 12-, and V alpha 24-specific monoclonal antibodies (mAbs), we estimated that between 50% and 99% of the CD25(+) Treg cells were dual specific, as compared with about 20% of their CD25(-) counterparts. Moreover, both TCRs were equally capable of transmitting signals upon ligation. Cells with 2 TCRs also expressed more FOXP3, the Treg-cell lineage specification factor, than cells with a single TCR. Our findings suggest that expression of 2 TCRs favors differentiation to the Treg-cell lineage in humans and raise the question of the potential functional consequences of dual specificity.