For unknown reasons, the eukaryotic transcription factor TFIID inefficiently recognizes promoters. Human TFIID was found to form highly specific homodimers that must dissociate before DNA binding. TFIID dimers formed through self-association of the TATA-binding polypeptide (TBP) subunit and could be immunoprecipitated with antibodies to TAF(II)250, the core subunit of TFIID. Chemical cross-linking experiments in HeLa cells revealed the presence of TBP dimers in vivo. These findings suggest that dimerization through TBP is the physiological state of TFIID when not bound to DNA. Thus, the inefficiency of TFIID binding to a promoter may be partly attributable to the competitive effect of dimerization.