The small protein p10/Ntf2p has been implicated in protein import in vitro (Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10212-10216; Paschal, B. M., and Gerace, L.(1995) J. Cell Biol. 129, 925-937). Here we present the first evidence that demonstrates an essential in vivo role for the NTF2 gene product in nuclear transport. The NTF2 locus was identified in a screen for temperature-sensitive Saccharomyces cerevisiae mutants defective in the localization of nuclear proteins. Genetic analysis demonstrates that the NTF2 gene is essential for viability in budding yeast. Two temperature-sensitive mutants, ntf2-1 and ntf2-2, that each contain single point mutations in highly conserved amino acid residues show defects in the localization of nuclear proteins but not in the export of poly(A)+ RNA following a shift to the nonpermissive temperature. An epitope-tagged version of Ntf2p was used to show that the protein is concentrated at the nuclear envelope. Finally, the human gene under the control of the yeast promoter fully substitutes for the deleted yeast gene. Taken together, these results demonstrate the exquisite functional conservation of this protein throughout evolution and indicate a critical in vivo role in nuclear transport.