Publisher Summary This chapter describes the gramicidin perforated patch technique and its application to CNS neurons. Gramicidin is a polypeptide antibiotic that forms small pores in the cell membrane similar to nystatin, but allows only the movement of monovalent cations excluding anions. The cation selectivity sequence of gramicidin in cell membranes is similar to that measured in lipid bilayers. Gramicidin is not permeable to Clˉ in lipid bilayers or in living cells. Thus, by adding gramicidin to the patch pipette solution, we can observe native responses from electrically and chemically excitable cells, while maintaining native [Clˉ]i, independently of the Clˉ concentration in the patch pipette solution. The application of gramicidin perforated patch to CNS neurons involves following steps: getting the patch, GABA responses, developmental change in glycine and GABA responses, effect of neuronal trauma, effect of H2O2 treatment, and effect of furosemide on intracellular [Clˉ]i.