The NEPH family comprises three transmembrane proteins of the Ig superfamily interacting with the glomerular slit diaphragm proteins podocin and ZO-1. NEPH1 binds to nephrin, another component of the slit diaphragm, and loss of either partner causes heavy proteinuria. NEPH2, which is strongly conserved among a large number of species, is also expressed in the kidney; however, its function is unknown. The authors raised NEPH2 antisera to demonstrate NEPH2 expression in a variety of mouse tissues, including the kidney and a podocyte cell line. The authors localized the expression of NEPH2 to the glomerular slit diaphragm by electron microscopy and show NEPH2 homodimerization and specific interactions with the extracellular domain of nephrin in vitro and in vivo. NEPH1, however, failed to interact with NEPH2. The authors detected immunoreactive NEPH2 in urine of healthy subjects, suggesting that the extracellular domain is cleaved under physiologic conditions. These findings were confirmed in vitro in podocyte cell culture. Shedding is increased by tyrosine phosphatase inhibitors and diminished by GM6001, an inhibitor of metalloproteinases. Overexpression experiments indicate an involvement of the MT1-matrix metalloproteinase. The results suggest a role for NEPH2 in the organization and/or maintenance of the glomerular slit diaphragm that may differ from the functions of NEPH1 and nephrin.