We previously reported cloning the rLot1 gene, and its human homolog (hLOT1), through analysis of differential gene expression in normal and malignant rat ovarian surface epithelial cells. Both human and rat ovarian carcinoma cell lines exhibited lost or decreased expression of this gene. Interestingly, the LOT1 gene localized at band q25 of human chromosome 6 which is a frequent site for LOH in many solid tumors including ovarian cancer. In this report we have further characterized the potential role of LOT1 in malignant transformation and developed evidence that the gene is a novel target of growth factor signaling pathway. Assays using transient transfections showed that LOT1 is a nuclear protein and may act as a transcription factor. In vitro and in vivo studies involving ovarian cancer cell lines revealed that expression of LOT1 is directly associated with inhibition of cellular proliferation and induction of morphological transformations. Additionally, we show that in normal rat ovarian surface epithelial cells Lot1 gene expression is responsive to growth factor stimulation. Its mRNA is strongly down-regulated by epidermal growth factor receptor (EGFR) ligands, namely EGF and TGF-alpha. Blocking the ligand-activated EGFR signal transduction pathway by the specific EGF receptor inhibitor, tyrphostin AG1478, and the MEK inhibitor, PD098059, restores the normal level of Lot1 gene expression. It appears that the regulation of Lot1 gene is unique to these ligands, as well as the growth promoting agent TPA, since other factors either did not affect Lot1 expression, or the effect was modest and transient. Altogether, the results suggest that Lot1 expression is primarily mediated via EGF receptor or a related pathway and it may regulate the growth promoting signals as a zinc-finger motif containing nuclear transcription factor.