The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.