The human homeobox-containing gene Hox 2.1 (hHox 2.1) and its murine cognate mHox 2.1 are part of evolutionarily conserved gene clusters, encode an identical Antennapedia-type homeodomain, and are expressed in a similar pattern in the developing embryo. We have isolated cDNA clones of hHox 2.1 and found that the human/murine Hox 2.1 gene structure is strikingly conserved, with only 3 out of 269 amino acid differences in the entire predicted protein sequence. We show that purified hHox 2.1 protein is a sequence-specific DNA binding protein capable of binding to a variety of DNA sequences, including multiple sites in the promoter of the hHox 2.1 gene. In a footprint titration assay, the apparent affinity of the hHox 2.1 protein for a consensus binding site (LP) increases when the site is present in tandem copies. Quantitative footprint challenge experiments revealed that the in vitro half-life of the protein-DNA complex is less than 30 s for a single LP binding site (kd greater than 1.4 min-1), but 126 min for proteins bound to two tandem LP binding sites (kd = 5.5 x 10(-3) min-1). A domain distinct from the homeodomain is necessary for cooperative DNA binding, because a 61-amino-acid peptide containing only the Hox 2.1 homeodomain can specifically bind to LP sites, but exhibits no cooperativity. A different full-length human homeodomain protein, hHox 1.3, was also found to show cooperative DNA binding quantitatively similar to hHox 2.1. Therefore, cooperative DNA binding to adjacent sites may be a crucial component in the overall affinity of mammalian Antennapedia-type homeodomain proteins for their DNA target sites.