Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase I␣ (CKI␣) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKI␣ and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKI␣-or Zw3-mediated Arm phosphorylaytion in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R؉ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKI␣ and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKI␣-mediated priming phosphorylation. Daxin stimulates Zw3-but not CKI␣-mediated Arm phosphorylation. Wg suppresses Zw3-but not CKI␣-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphoylation is due to CKI␣ and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.