Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl- ([Cl-i]) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by: 1) two-photon imaging of the Cl- sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl- sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs), but were progressively ...