LIS1 is a product of the causative gene for type I lissencephaly characterized by a smooth brain surface due to a defect in neuronal migration during brain development and a regulatory subunit of platelet-activating factor acetylhydrolase (PAF-AH). It is also a mammalian homologue of the fungal nuclear distribution (nud) gene, nudF, which controls the migration of fungal nuclei. Using the two-hybrid system, we identified a novel LIS1-interacting protein, rat NUDE (rNUDE), and found that it is a mammalian homologue of another fungal nud gene product, NUDE, and Xenopus mitotic phosphoprotein 43 which is phosphorylated in a cell cycle-dependent manner. rNUDE and the catalytic subunits of PAF-AH interact with the N- and C-termini of LIS1, respectively. However, these proteins, instead of simultaneously binding to LIS1, appeared to bind to LIS1 in a competitive manner. These results suggest that LIS1 functions in nuclear migration by interacting with multiple intracellular proteins in mammals.