A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.