Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.