Retinal ganglion cells exhibit fast and slow inhibitory synaptic glycine currents that can be selectively inhibited by strychnine and 5,7-dichlorokynurenic acid (DCKA), respectively. In this study we examined whether strychnine and DCKA selectivity correlated with the subunit composition of the glycine receptor. Homomeric alpha1, alpha2 or alpha2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the alpha1 subunit, responses to 200 microm glycine were blocked by 1 microm strychnine but not by 500 microm DCKA. In cells expressing the alpha2 subunit, both 1 microm strychnine and 500 microm DCKA were effective antagonists of 200 microm glycine. In cells expressing alpha2* subunits, which are much less glycine-sensitive, 10 mm glycine was inhibited by 500 microm DCKA but not by 1 microm strychnine. A single amino acid mutation in the alpha1 subunit (R196G), converted this subunit from DCKA-insensitive to DCKA-sensitive. In conclusion, the comparative effectiveness of strychnine and DCKA can be used to distinguish between the alpha1, alpha2 and alpha2* receptor responses. Furthermore, a single amino acid near the glycine receptor's putative agonist binding site may account for differences in DCKA sensitivity amongst the alpha subunits.