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Holt–Oram syndrome is caused by mutations in TBX5, a member of the T-box gene family. In order to identify DNA sequences to which the TBX5 protein binds, we have performed an in vitro binding site selection assay. We have identified an 8 bp core sequence that is part of the Brachyury consensus-binding site. We show that TBX5 binds to the full palindromic Brachyury binding site and to the half-palindrome, whereas Brachyury does not bind to the TBX5 site. Amino acids 1–237 of TBX5 are required for DNA binding. Analysis of the effects of specific substitution mutations that arise in Holt–Oram patients indicates that G80R and R237Q eliminate binding to the target site. DNA database analysis reveals that target sites are present in the upstream regions of several cardiac-expressed genes including cardiac α actin, atrial natriuretic factor, cardiac myosin heavy chain α, cardiac myosin heavy chain β, myosin light chain 1A, myosin light chain 1V and Nkx2.5. Cell transfection studies demonstrate that TBX5 activates the transcription of an atrial natriuretic factor reporter construct and this effect is significantly reduced by deletion of the TBX5 binding site.