Semliki Forest virus nonstructural protein nsP2, when expressed in the absence of other viral proteins, is transported into the nucleus, with a specific enrichment in nucleoli. This implies that information for its nuclear targeting must be in nsP2 itself. To define the nuclear and nucleolar targeting signals of nsP2, a series of in-frame deletions were made in its coding sequence. We have identified one amino-terminal (residues 25-110) and another carboxy-terminal (residues 634-661) deletion, both of which rendered nsP2 cytoplasmic. The carboxy-terminal region had a pentapeptide sequence P 648R RRV, which resembles some of the known nuclear localization signals. When the three arginines were substituted with aspartic acids, the mutant nsP2 was localized in the cytoplasm. Especially arginines 648-649 were shown to be critical. The carboxy-terminal 232 amino acid residues of nsP2 were able to translocate beta-galactosidase to the nucleus as a fusion protein, whereas the amino-terminal 110 residues failed to do so. Studies with different beta-galactosidase-nsP2 fusions showed that the pentapeptide is necessary for nuclear transport but that its activity is strongly affected by the context. The deletion analysis indicated also that the nuclear localization signal was not sufficient for nucleolar targeting of nsP2. The nucleolar targeting sequence of nsP2 was tentatively mapped between residues 470 and 539, and at least residues 470-489 are an essential part of it.