It is now a well-accepted view that cation-driven Cl(-) transporters in neurons are involved in determining the intracellular Cl(-) concentration. In the present review, we propose that additional factors, which are often overlooked, contribute substantially to the Cl(-) gradient across neuronal membranes. After briefly discussing the data supporting and opposing the role of cation-chloride cotransporters in regulating Cl(-), we examine the participation of the following factors in the formation of the transmembrane Cl(-) gradient: (i) fixed 'Donnan' charges inside and outside the cell; (ii) the properties of water (free vs. bound); and (iii) water transport through the cotransporters. We demonstrate a steep relationship between intracellular Cl(-) and the concentration of fixed negative charges on macromolecules. We show that in the absence of water transport through the K(+)-Cl(-) cotransporter, a large osmotic gradient builds at concentrations below or above a set value of 'Donnan' charges, and show that at any value of these fixed charges, the reversal potential for Cl(-) equates that of K(+). When the movement of water across the membrane is a source of free energy, it is sufficient to modify the movement of Cl(-) through the cotransporter. In this scenario, the reversal potential for Cl(-) does not closely follow that of K(+). Furthermore, our simulations demonstrate that small differences in the availability of freely diffusible water between inside and outside the cell greatly affect the Cl(-) reversal potential, particularly when osmolar transmembrane gradients are minimized, for example by idiogenic osmoles. We also establish that the presence of extracellular charges has little effect on the chloride reversal potential, but greatly affects the effective inhibitory conductance for Cl(-). In conclusion, our theoretical analysis of the presence of fixed anionic charges and water bound on macromolecules inside and outside the cell greatly impacts both Cl(-) gradient and Cl(-) conductance across neuronal membranes.