Ecdysone receptor (EcR) and its heterodimer, ultraspiracle protein (USP), are ligand dependent transcriptional factors. Transcriptional factors are often regulated by phosphorylation. Protein kinase consensus recognition sequence analysis of Drosophila EcR and USP revealed putative phosphorylation sites for protein kinase C (PKC) and casein kinase II (CKII). By using specific protein kinase inhibitors, we have shown that PKC, not CKII, is responsible for EcR and USP phosphorylation. Inhibition of the EcR/USP phosphorylation by protein kinase inhibitors resulted in the inhibition of a specific set of 20E-response gene expression.