This paper reports the identification of a Rho family nucleotide exchange factor termed mNET1 as a candidate-interacting partner for the first PDZ domain of MAGI-1, a membrane-associated guanylate kinase with inverted arrangement of protein-protein interacting modules, mNET1 was identified in a yeast two-hybrid screen and has a consensus tripeptide for PDZ domain binding at its extreme carboxy-terminus. In addition to this sequence, a cluster of basic residues located near the carboxy terminus is essential for the binding. The interaction of the first PDZ domain of MAGI-1 with mNET1 was documented using a variety of biochemical methods.