Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr²⁴, Ser²⁵⁶ and Ser³¹⁹) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser³²⁹) as a novel in vivo phosphorylation site on FKHR. Ser³²⁹ phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser³²⁹ is phosphorylated in unstimulated HEK-293cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser³²⁹ specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser³²⁹in vivo.