Neuroplastin-65 (Np65) is a brain-specific cell adhesion molecule belonging to the immunoglobulin superfamily. Homophilic trans-interaction of Np65 mediates adhesion between cells and modulates synaptic plasticity. This interaction solely occurs through the first immunoglobulin (Ig) module of Np65, but the exact binding mechanism has not yet been elucidated. In this study, we identify the homophilic binding motif of Np65 and show that a synthetic peptide modeled after this motif, termed enplastin, binds to Np65. We demonstrate that both Np65- and enplastin-induced intracellular signaling depends on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro and on the initial phase of spatial learning in rats.