Lithium is a commonly prescribed mood-stabilizing drug. However, chronic treatment with lithium induces numerous kidney-related side effects, such as dramatically reduced aquaporin 2 (AQP2) abundance, altered renal function, and structural changes. As a model system, inner medullary collecting ducts (IMCD) isolated from rats treated with lithium for either 1 or 2 weeks were subjected to differential 2D gel electrophoresis combined with mass spectrometry and bioinformatics analysis to identify (i) signaling pathways affected by lithium and (ii) unique candidate proteins for AQP2 regulation. After 1 or 2 weeks of lithium treatment, we identified 6 and 74 proteins with altered abundance compared with controls, respectively. We randomly selected 17 proteins with altered abundance caused by lithium treatment for validation by immunoblotting. Bioinformatics analysis of the data indicated that proteins involved in cell death, apoptosis, cell proliferation, and morphology are highly affected by lithium. We demonstrate that members of several signaling pathways are activated by lithium treatment, including the PKB/Akt-kinase and the mitogen-activated protein kinases (MAPK), such as extracellular regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38. Lithium treatment increased the intracellular accumulation of beta-catenin in association with increased levels of phosphorylated glycogen synthase kinase type 3beta (GSK3beta). This study provides a comprehensive analysis of the proteins affected by lithium treatment in the IMCD and, as such, provides clues to potential lithium targets in the brain.