The TATA box-binding protein (TBP) has a fundamental role in eukaryotic cell metabolism, since it is necessary for transcription of class I, class II, and class III genes; in fact, TBP is the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB. Contrary to the previously hypothesized existence of a family of genes coding for DNA-binding proteins highly related to TBP, our experiments show that the segment coding for the evolutionarily well-conserved carboxyl-terminal domain, involved in DNA binding, is unique; accordingly, we conclude that the TBP locus itself, which we have localized to 6q27, is single copy in the human genome. On the other hand, a cDNA fragment coding for the evolutionarily variable amino-terminal domain detects multiple cross-hybridizing sequences in the genome of higher eukaryotes. We suggest that the common motif is represented by the long string of glutamine codons, which characterizes the amino-terminal segment of human TBP: in fact, other proteins involved in transcription, such as TAF II 110, Sp1, and some homeobox proteins, are known to contain glutamine-rich segments.