The tuberous sclerosis complex 1 and 2 (TSC1 and TSC2) genes and their products hamartin and tuberin, respectively, are involved in several regulatory cellular mechanisms, including the mTOR pathway that is important for proliferation, cell migration, and protein synthesis. In addition, it has been suggested that this pathway plays a role in regulating Na+-K+-2Cl- and K+-Cl- cotransporters (NKCC and KCC, respectively) [41, 42, 43]. Therefore, the TSC1 and TSC2 genes are expected to play a significant role in regulating cell volume, and K+ and Cl- homeostasis. This study tests the hypothesis that the TSC1 and TSC2 genes, which control cell processes through the mTOR pathway, are important for the function of the Na+/K+ pump (NKP), Na+,K+,2Cl- and K+,2Cl- cotransporters (KCC and KCC, respectively) and for the maintenance of intracellular K+ (Ki ) content in mouse embryonic fibroblasts (MEF) cells. The cellular models are wild type (WT) and knockouts (KO) of the TSC1 or TSC2 genes, TSC1-KO and TSC2-KO, respectively. K+ homeostasis was assessed through the activities of NKP, NKCC and KCC determined by rubidium (Rb+, a K+ congener) influx and Ki content in the presence and absence of the NKP-inhibitor ouabain, the NKCC-inhibitor bumetanide and by Cl--dependent Rb+ influx via KCC. Optimal ouabain and bumetanide concentrations were obtained from the dose-response curves of Rb+ influx and Ki content inhibition toward these compounds. In support of the hypothesis, the main findings indicate that absence of the TSC1, and especially, the TSC2 gene, which are associated with depression of the mTOR pathway [4, 5, 8], leads to accelerated cell proliferation and migration, and abnormal activities of all the transporters tested revealing the importance of the TSC1 and TSC2 gene products, hamartin and tuberin, respectively, in cellular K+ homeostasis in MEF cells. Moreover, abnormalities in ion transport of TSC2-KO cells is commensurate with the pathological manifestation and incidence of tuberous sclerosis complex in the human population. These findings also uncover potential targets for more efficient therapy.