The tuberous sclerosis-2 (Tsc-2) gene is a suppressor of renal tumorigenesis, and an early target of reactive oxygen species induced renal cancer. Tuberin, the protein product of the Tsc-2 gene, participates in the regulation of cell proliferation, although the mechanism by which it suppresses proliferation is unknown. QT-RRE cell lines, derived from quinol-thioether-transformed primary renal epithelial cells from Eker rats, lack tuberin expression due to loss of heterozygosity of the Tsc-2 gene. These cell lines were utilized to examine the mechanism by which tuberin exerts its anti-proliferative action. Loss of tuberin function correlates with high extracellular signal-regulated kinase activity (ERK) (Yoon, et al., 2002), which could contribute to the formation of renal tumors. In this study we sought to identify possible downstream effectors regulated by tuberin, utilizing QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. Constitutively high ERK, B-Raf and Raf-1 activities were observed in QT-RRE cells. However, restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA significantly decreased both ERK and B-Raf activity, without significant changes in Raf-1 activity, suggesting tuberin functions as an upstream negative regulator of the ERK pathway. High ERK activity was not mediated through EGF receptor activation, but treatment with genistein demonstrated that protein kinases are involved in ERK cascade activation. The data indicate that loss of tuberin results in the up-regulation of the ERK signaling pathway with subsequent increases in new DNA synthesis, and ultimately, tumor formation.