Simultaneous recording of changes in intracellular Cl- concentration ([Cl-]i) in individual neurons situated in different layers (e.g. II/III-VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy-N-ethylquinolinium iodide (MEQ). Gamma-aminobutyric acid (GABA) caused a measurable increase in [Cl-]i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl- pump and cation-Cl- co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl- and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at > 30 degrees C. Estimation of [Cl-]i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl- concentrations ([Cl-]) in the presence of tributyltin, a Cl(-)-OH- antiporter, nigericin, a K+-H+ antiporter, and KSCN. This enables comparison of [Cl-]i between neurons in different slices. Thus, optical imaging of [Cl-]i in brain slices can provide valuable spatial information about [Cl-]i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.