OBJECTIVE Investigate the effects of diazepam (DZP) on γ2 subunit containing GABA type A receptor (GABAAR) trafficking. METHODS Immunofluorescence microscopy measured surface GABAARs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol · L- 1 DZP. Biochemical studies of mice injected with 10 mg·kg-1 DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post- injection. Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L- 1 DZP exposure. A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein (γ2pHFAP) measured lysosomal targeting of γ2 containing GABAARs. RFP-gephyrin and γ2pHFAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching (FRAP). RESULTS Extrasynaptic levels of γ2 GABAARs decreased by 12.2%, while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control (P<0.05). Dendritic levels of gephyrin were also reduced to 74.1% of control, while S270 phosphorylation was elevated by 25.2% (P<0.05; P<0.01). Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels, respectively (P<0.05; P<0.01). 12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control (P<0.05). Internalized γ2pHFAP GABAARs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control. Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP. CONCLUSION DZP treatment decreases γ2 GABAAR levels and gephyrin scaffolding function after one day of exposure, which may contribute to the formation of DZP tolerance.