Nucleolar shuttle protein B23 was found to bind to human immunodeficiency virus protein Tat, and this binding required the nucleolar localization motif of Tat. A fusion protein containing the B23 binding domain and beta-galactosidase caused mislocalization of Tat to the cytoplasm and inhibited the transactivation activity of Tat. These data suggest that B23 is a human factor necessary for the nucleolar localization of Tat.