AbstractPrimary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1–11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled‐coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences (CTSs) are unknown. In the present study, we generated a series of GFP‐tagged deletion constructs of Nphp3 and tried to find the CTSs of Nphp3. We found that the N‐terminal 201 amino acid fragment (Nphp3 [1–201]), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N‐terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N‐terminal fragments, Nphp3 (8–201), Nphp3 (52–201), and Nphp3 (96–201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two‐step process. © 2012 Wiley Periodicals, Inc.