Ginkgolides, active constituents of Ginkgo biloba extracts, potently block the glycine receptor chloride channel (GlyR). Ginkgolides A, B, C and J are structurally similar, varying only by the presence or absence of oxygens at their R1 and R2 positions. The aim of this study was to understand how variable ginkgolide groups bind to pore-lining 2' and 6' residues in the alpha1 GlyR. Ginkgolide potency was not affected by G2'A or G2'S mutations, suggesting 2' residues are not important for ginkgolide coordination. Analysis of the alpha1(T6'S) GlyR suggests that ginkgolides bind to this receptor via hydrogen bonds between T6'S and ginkgolide R1 hydroxyls. The abolition of block by the T6'A and T6'V mutations but not by the T6'S mutation implies the existence a second transmembrane domain alpha-helical kink formed by hydrogen bonding between 6' threonine and serine sidechains and backbone carbonyl oxygens. We also found that ginkgolide A binds in different orientations in the closed and open states of a mutant GlyR, possibly reflecting its enhanced flexibility relative to other ginkgolides. Together these results indicate that small variations in ginkgolide structure or pore structure can lead to drastic potency variations. This property may be exploited to create improved pharmacological probes for discriminating among anionic Cys-loop receptor isoforms with 6' structural variations.