Ecdysone receptor (EcR) and its heterodimer, ultraspiracle protein (USP), are ligand-dependent transcriptional factors that mediate the action of molting hormone 20-hydroxyecdysone. The activities of transcriptional factors are subjected to regulation not only by transcriptional/translational mechanisms, but also by posttranslational mechanisms such as phosphorylation. Protein kinase consensus recognition sequence analysis of Drosophila EcR and USP reveals multiple phosphorylation sites for protein kinase C (PKC) and casein kinase II (CKII) on EcR and USP sequence. By using specific protein kinase inhibitors, we have shown that PKC, not CKII, is responsible for USP phosphorylation. Inhibition of PKC activity by protein kinase inhibitors blocked USP phosphorylation, resulting in inhibition of 20E-induced gene expression at both transcriptional and translational levels. The composite data suggest that PKC-mediated USP phosphorylation is required for 20E-induced gene expression in the salivary glands of Drosophila melanogaster.