Contrary to its effect on the [gamma]–aminobutyric acid type A and C receptors, picrotoxin (PTX) antagonism of the a₁ homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and non-selective between the PTX components picrotoxinin and picrotin. PTX antagonism of the embryonic a₂ homomeric GlyR is known to be use-dependent, reflecting a channel-blocking mechanism, but the selectivity of PTX antagonism of the embryonic a₂ homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch-clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of a₂ homomeric GlyRs stably transfected into Chinese hamster ovary (CHO) cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents in a concentration-dependent manner, picrotin had a 30 times lower efficiency than picrotoxinin. Competitive-like and noncompetitive mechanisms were observed for picrotin and picrotoxinin. Picrotin-evoked inhibition displayed voltage-dependency, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these PTX components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but could both be trapped at their binding site when the channel closed during glycine dissociation. Based on these observations we propose kinetic Markov models in which picrotoxinin and picrotin bind to the open state and the fully liganded closed state. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking a₂ homomeric GlyR.