The gamma 2 subunit is required for benzodiazepine modulation of the GABA(A) receptor. Alternate splicing of precursor GABA(A) gamma 2 mRNA results in two splice variants, a short (gamma 2S) and a long (gamma 2L) variant. We investigated the roles of these splice variants in benzodiazepine pharmacology using mice lacking genes for the gamma 2L splice variant. Sleep time responses to midazolam and zolpidem were 20 and 18% greater, respectively, in null allele mice compared with wild-type mice, while responses to nonbenzodiazepine agents such as etomidate and pentobarbital were unchanged. Although the GABA(A) receptor number was not altered in null allele mice, there was a corresponding increase in affinity of brain membranes for benzodiazepine agonists (midazolam, diazepam, and zolpidem), while affinity for benzodiazepine inverse agonists (beta CCM and Ro15-4513) was decreased. These changes were not observed in inbred mice of the parental strains (C57BL/6J and 129/SvJ) used to create the genetically altered mice, indicating that differences between gamma 2L null allele and wild-type mice were unlikely to be simply due to cosegregation of linked alleles. Absence of the gamma 2L splice variant increases the affinity of receptors for benzodiazepine agonists, and is associated with a modest increase in behavioral sensitivity to benzodiazepine agonists. Lack of the gamma 2L subunits may shift the GABA(A) receptor from an inverse agonist-preferring toward an agonist-preferring configuration.