Neocortical neurons mediate the sedative and anticonvulsant properties of benzodiazepines. These agents enhance synaptic inhibition via positive modulation of γ-aminobutyric acid (GABAA) receptors harboring α1-, α2-, α3- or α5-protein subunits. Benzodiazepine-sensitive GABAA receptors containing the α5-subunit are abundant in the neocortex, but their impact in controlling neuronal firing patterns is unknown. Here we studied how the discharge rates of cortical neurons are modified by a positive (SH-053-2′F-R-CH3) and a negative (L 655,708) α5-subunit-preferring allosteric modulator in comparison to diazepam, the classical non-selective benzodiazepine. Drug actions were characterized in slice cultures from wild-type and α5(H105R) knock-in mice by performing extracellular multi-unit-recordings. In knock-in mice, receptors containing the α5 subunit are insensitive to benzodiazepines. The non-selective positive allosteric modulator diazepam decreased the discharge rates of neocortical neurons during episodes of ongoing neuronal activity (up states). In contrast to diazepam, the α5-preferring positive modulator SH-053-2′F-R-CH3 accelerated action potential firing during up states. This promoting action was absent in slices from α5(H105R) mice, confirming that it is mediated by the α5-subunit. Consistent with these observations, the negative α5-selective modulator L 655,708 inhibited up state action potential activity in slices from wild-type mice. The opposing actions of diazepam and SH-053-2′F-R-CH3, which both enhance GABAA receptor function but differ in subtype-selectivity, uncovers contrasting roles of GABAA receptor subtypes in controlling the firing rates of cortical neurons. These findings may have important implications for the design of novel anaesthetic and anticonvulsant benzodiazepines displaying an improved efficacy and fewer side effects.