The effect of picrotoxinin on glycine-induced chloride currents was studied in dissociated rat hippocampal neuron culture in whole-cell and excised outside-out patches. Picrotoxinin blocked the glycine induced chloride currents. The picrotoxinin effect at 20 microM on glycine dose response relationship suggested a competitive mechanism. However, at 1 mM, the picrotoxinin effect was largely noncompetitive. In excised patches, glycine activated two types of channels distinguished by a difference in conductances. The first group had single channel conductances of around 47 pS and another around 100 pS. Occasionally, both types of channels were found in the same excised patch. Low concentration of picrotoxinin selectively blocked large conductance channels. At higher concentrations of 0.5 to 1 mM, picrotoxinin blocked the small conductance channels by a flickering block. These findings indicate that the whole-cell glycine current in rat hippocampal neurons is mediated by at least two types of channels. The two types of channels have distinct conductance, picrotoxinin sensitivity and different mechanism of picrotoxinin block.