NORE1A is a growth and tumor suppressor that is inactivated in a variety of cancers. NORE1A has been shown to bind to the active Ras oncogene product. However, the mechanism of NORE1A-induced growth arrest and tumor suppression remains unknown. Using anchorage-independent growth assays, we mapped the NORE1A effector domain (the minimal region of the protein responsible for its growth-suppressive effects) to the fragment containing the central and Ras association domains of NORE1A (amino acids 191-363). Expression of the NORE1A effector domain in A549 lung adenocarcinoma cells resulted in the selective inhibition of signal transduction through the ERK pathway. The full-length NORE1A (416 amino acids) and its fragments capable of growth suppression were localized to centrosomes and microtubules in normal and transformed human cells in a Ras-independent manner. A mutant that was deficient in binding to centrosomes and microtubules was also deficient in inducing cell cycle arrest. This suggests that cytoskeletal localization is required for growth-suppressive effects of NORE1A. Ras binding function was required for growth-suppressive effects of the full-length NORE1A but not for the growth-suppressive effects of the effector domain. Our studies suggest that association of NORE1A with cytoskeletal elements is essential for NORE1A-induced growth suppression and that the ERK pathway is a target for NORE1A growth-suppressive activities.