Ecdysone receptor (EcR) and its heterodimer, ultraspiracle protein (USP), are ligand dependent transcriptional factors. Transcriptional factors are often regulated by phosphorylation. Although EcR and USP have been shown to be phosphoproteins, the role of EcR and USP phosphorylation in mediating EcR and USP translocation and 20E-induced gene expression remains unclear. By using specific protein kinase and protein phosphatase inhibitors, the effect of protein phosphorylation on EcR and USP translocation and 20E-induced gene expression in Drosophila melanogaster was investigated and discussed.