Cep135 is a 135-kDa, coiled-coil centrosome protein important for microtubule organization in mammalian cells [Ohta et al., 2002: J. Cell Biol. 156: 87-99]. To identify Cep135-interacting molecules, we screened yeast two-hybrid libraries. One clone encoded dynamitin, a p50 dynactin subunit, which localized at the centrosome and has been shown to be involved in anchoring microtubules to centrosomes. The central domain of p50 binds to the C-terminal sequence of Cep135, this was further confirmed by immunoprecipitation and immunostaining of CHO cells co-expressing the binding domains for Cep135 and p50. Exogenous p50 lacking the Cep135-binding domain failed to locate at the centrosome, suggesting that Cep135 is required for initial targeting of the centrosome. Altered levels of Cep135 and p50 by RNAi and protein overexpression caused the release of endogenous partner molecules from centrosomes. This also resulted in dislocation of other centrosomal molecules, such as γ-tubulin and pericentrin, ultimately leading to disorganization of microtubule patterns. These results suggest that Cep135 and p50 play an important role in assembly and maintenance of functional microtubule-organizing centers. Cell Motil.