CD47 and SHPS-1 are transmembrane proteins that interact with each other through their extracellular regions and constitute a bidirectional cell-cell communication system (the CD47-SHPS-1 system). We have now shown that the trans-interaction of CD47 and SHPS-1 that occurred on contact of CD47-expressing CHO cells and SHPS-1-expressing CHO cells resulted in endocytosis of the ligand-receptor complex into either cell type. Such trans-endocytosis of CD47 by SHPS-1-expressing cells was found to be mediated by clathrin and dynamin. A juxtamembrane region of SHPS-1 was indispensable for efficient trans-endocytosis of CD47, which was also regulated by Rac and Cdc42, probably through reorganization of the actin cytoskeleton. Inhibition of trans-endocytosis of CD47 promoted the aggregation of CD47-expressing cells with the cells expressing SHPS-1. Moreover, CD47 expressed on the surface of cultured mouse hippocampal neurons was shown to undergo trans-endocytosis by neighboring astrocytes expressing endogenous SHPS-1. These results suggest that trans-endocytosis of CD47 is responsible for removal of the CD47-SHPS-1 complex from the cell surface and hence regulates the function of the CD47-SHPS-1 system, at least in neurons and glial cells.