Nucleolar localization of the fusion protein of rat ribosomal protein L31 and beta-galactosidase was investigated by immunocytochemical means, using the ribosomal protein deletion or substitution mutants that were transiently expressed in COS-1 cells. The signal responsible for nucleolar localization is encoded by the amino acid residues 87 to 92, RLSRKR, located near the C terminus of the ribosomal protein. Mutation of residues 87R and 90R prevented nucleolar localization, whereas mutations including 90R prevented nuclear and nucleolar localization. Further mutations in the sequence revealed that the tetrapeptide RLSR, which was amenable to substitutions at the L and S positions, is critical for nucleolar localization.