Formation of anterior structures in the Drosophila embryo requires the product of the gene bicoid. The bicoid protein contains a homeodomain and may exert its effects in early development by regulating transcription of the gap gene, hunchback (hb). Consistent with this view, we have demonstrated that DNA-bound Bicoid fusion proteins stimulate gene expression. We used the gene activation phenotype in yeast to study DNA recognition by the Bicoid homeodomain. We found that a single amino acid replacement at position 9 of the recognition helix was sufficient to switch the DNA specificity of the Bicoid protein. The altered specificity Bicoid mutants recognized DNA sites bound by Ultrabithorax, fushi tarazu, and other related homeo-domain proteins. Our results suggest that DNA specificity in Bicoid and Antennapedia class proteins is determined by recognition helix residue 9.