The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His(2)-Cys(2) zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a "blocking" domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Delta mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.