Single-channel currents through GABA- and glycine-activated chloride channels of post-natal tissue-cultured hippocampal neurons were measured to determine their anion selectivity and their concentration dependence of permeation. Current-voltage relations for both agonists displayed rectification with single-channel conductance increasing at positive potentials. Permeabilities determined from reversal potentials were maximal for anions with a diameter of about 4 A. Larger diameter anions had lower permeabilities, consistent with an approximate pore diameter of 6 A for both agonist-activated channels. The permeability for anions of similar size was greatest for those ions with a more symmetrical charge distribution (e.g. NO3- > Bicarbonate-). The permeability sequence was SCN- > NO3- > I- > Br- > Cl- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-, whereas the conductance sequence for anion efflux was Cl- > Br- > NO3- > I- > SCN- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-. These results suggest that the ions interact with sites within the channel, with hydration forces contributing an important component to the barrier for ion entry into the channel. The spherically symmetrical halides displayed an exponential relation between relative permeability and hydration energy. Concentration dependence of conductance for Cl- channels in symmetrical Cl- solutions with agonist in the pipette showed an increase at positive potentials and a decrease at negative potentials. GABA- and glycine-activated channels also exhibited anomalous mole-fraction effects in a mixture of Cl- and SCN-. These results suggest that both agonist-activated channels act as multi-ion pathways and have similar permeation characteristics.