Semaphorin 3A (Sema3A) is a member of semaphorins and functions as an axonal repulsive guidance molecule. Neuropilin-1 and plexin-As form receptor complexes for Sema3A and plexin-As are thought to initiate the intracellular signaling cascade. However, the molecule by which plexin-As transduce their signal is not well understood. We searched molecules that interact with intracellular domains of plexin-A1 by yeast two-hybrid screening and identified a 349 amino acid fragment of plexin-B1 as a plexin-A1 interacting protein. We, then, cloned mouse plexin-B1 and confirmed their interaction in a mammalian expression system. Plexin-B1 physically associated with plexin-A1, but not with plexin-A2 or A3. Northern blot analysis showed the expression of both plexin-A1 and B1 in adult brain. We propose that plexin-A1 and B1 interact in the adult brain and transduce Sema3A signaling in cooperation.