The antibiotic gramicidin, when incorporated into lipid membranes, forms pores that are exclusively permeable to monovalent cations and small unchanged molecules. We report the use of gramicidin for perforated patch-clamp recordings in the whole-cell mode. Recordings were performed in cultured rat spinal cord dorsal horn neurons. Cells had stable resting potentials and series resistances for times routinely exceeding 60 min. To test if intracellular chloride concentration ([Cl]i) remains stable with this technique, we measured responses to agonists of glycine and GABAA receptors, both of which gate chloride conductances. The driving force for these responses remained stable at values that differed significantly from values that would be expected if [Cl-]i were biased towards pipette [Cl-]. We conclude that gramicidin perforated-patch recording, in addition to other properties of the perforated-patch recording technique, has the advantage of not altering [Cl-]i. It is, therefore, an electrophysiological method particularly suitable for studies of anionic channels when [Cl-]i is a variable of interest, as well as for studies of homeostatic [Cl-]i regulation.