The minimal T-cell receptor (TCR) β-chain (TCRβ) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4β-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR Vβ promoter decamer or to the immunoglobulin enhancer κE2 or μEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.