We present evidence that the in vivo activity of the HOX protein Antennapedia (ANTP) is modified because of phosphorylation by the serine/threonine kinase casein kinase II (CKII). Using an in vivo assay a form of ANTP that has alanine substitutions at its CKII target sites has, in addition to wild-type ANTP functions, the ability to alter severely thoracic and abdominal development. The novel functions of this protein suggest that this form of ANTP is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of ANTP that contains acidic amino acid substitutions at its CKII target sites, thereby mimicking a constitutively phosphorylated ANTP protein, is greatly reduced. This hypoactive form of ANTP, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. Our results suggest that phosphorylation of ANTP by CKII is important for preventing inappropriate activities of this homeotic protein during embryogenesis.