Using an in vivo assay for segment identity, the structural differences that distinguish two Drosophila homeotic selector proteins, Ultrabithorax (Ubx) and Antennapedia (Antp), have been investigated. There are at least two independent parts of Ubx and Antp that contribute to their functional specificities: (1) their homeo domains and (2) residues carboxy-terminal to their homeo domains (C-tails). In the absence of any C-tail, differences in 5 homeo domain amino acids are sufficient to distinguish between the functions of Ubx and Antp. Two of these are at the amino terminus of the homeo domain and could contact DNA directly. A three dimensional model suggests that the other 3 homeo domain residues and the C-tails are unlikely to contact DNA. In addition, we demonstrate that the assay used to measure the segment identity functions of Ubx and Antp is independent of any homeotic selector gene normally active in thoracic and abdominal segments. Therefore, it is likely that this assay measures the coordinate regulation of many downstream target genes. This expectation is confirmed for at least one Ubx target gene, Distal-less.