The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-1 cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This con®guration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunode®ciency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the ®rst NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three dierent backbones with RPS25 cDNA fragments. Immuno¯uorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/ nucleolar targeting signal. The identi®ed peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNAbinding transactivators.