Mutations in APC or in beta-catenin, which are common in colon cancer, lead to constitutive activation of beta-catenin/Tcf-dependent signaling. alpha-Catenin is also found in some colon cancer cell nuclei, and loss of its expression correlates with increased beta-catenin/Tcf transcriptional activity. Moreover, targeted expression of alpha-catenin in the nucleus inhibits beta-catenin/Tcf-dependent transcription. Thus, an understanding of the regulation of alpha-catenin localization could provide insight into the control of beta-catenin signaling. While the beta-catenin/Tcf complex can promote nuclear import of alpha-catenin, the mechanism for its nuclear export is not known. We found that leptomycin B (LMB) inhibited nuclear export of GFP-alpha-catenin in HCT116 colon cancer cells, suggesting that alpha-catenin localization is regulated by CRM-1-dependent nuclear export. We identified two putative nuclear export signals in a domain of alpha-catenin that overlaps with the beta-catenin binding domain. Using a nuclear export assay, we determined that one of these (NES1) is a weak LMB-insensitive NES, whereas the other (NES2) is strong and LMB-sensitive. Mutations in either NES reduced nuclear export of alpha-catenin in HCT116 cells. In addition, mutations in NES1, but not NES2, reduced binding of alpha-catenin to beta-catenin and impaired the ability of alpha-catenin to repress beta-catenin/Tcf-dependent transcription. Therefore, NES1 is required both for repression of beta-catenin signaling and for nuclear export, while NES2 is required only for nuclear export.