Intracellular chloride activity measurements were obtained from mudpuppy retinal neurons using dual microelectrodes, one of which was made chloride-selective by filling the tip with chloride liquid ion exchange resin. In addition ionic substitution experiments were carried out in the perfused retina-eyecup preparation of the mudpuppy. A comparison of the membrane potential and calculated chloride equilibrium potential shows that retinal neurons differ in relative transmembrane chloride distribution. Ganglion cells have an ECl more negative than the resting membrane potential, whereas amacrine cells have a passive distribution of chloride. Horizontal cells have chloride distributed such that an increase in chloride conductance is depolarizing. On-bipolars have a chloride distribution similar to that of horizontal cells whereas off-bipolars show either passive distribution or some chloride accumulation. These findings are consistent with the idea that chloride ions may play a role as a depolarizing driving force for electrogenic activity of horizontal cells and on-bipolars. Results with Cl substitution experiments are consistent with this interpretation.