CLIP-170/Restin belongs to a family of conserved microtubule (MT)-associated proteins, which are important for MT organization and functions. CLIP-170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP-170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12-rapamycin-associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP-170. CLIP-170 is phosphorylated in vivo at multiple sites, including rapamycin-sensitive and -insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin-sensitive sites. In addition, rapamycin inhibited the ability of CLIP-170 to bind to MTs. Our observations suggest that multiple CLIP-170 kinases are involved in positive and negative control of CLIP-170, and FRAP is a CLIP-170 kinase positively regulating the MT-binding behavior of CLIP-170.