AbstractCortical activity patterns are strongly modulated by fast synaptic inhibition mediated through ionotropic, chloride-conducting receptors. Consequently, chloride homeostasis is ideally placed to regulate activity. We therefore investigated the stability of baseline [Cl^-]_i in adult mouse neocortex, using in vivo two-photon imaging. We found a two-fold increase in baseline [Cl^-]_i in layer 2/3 pyramidal neurons, from day to night, with marked effects upon both physiological cortical processing and seizure susceptibility. Importantly, the night-time activity can be converted to the day-time pattern by local inhibition of NKCC1, while inhibition of KCC2 converts day-time [Cl^-]_i towards night-time levels. Changes in the surface expression and phosphorylation of the cation-chloride cotransporters, NKCC1 and KCC2, matched these pharmacological effects. When we extended the dark period by 4 h, mice remained active, but [Cl^-]_i was modulated as for animals in normal light cycles. Our data thus demonstrate a daily [Cl^-]_i modulation with complex effects on cortical excitability.