Saccharomyces cerevisiae nuclear proteins are shown to localize specifically to isolated yeast nuclei under conditions selective for nuclear proteins. Nuclear association is time- and temperature-dependent, requires ATP hydrolysis, and is abolished by protease pretreatment of nuclei. The nucleus-localized protein is translocated across the nuclear envelope as determined by inaccessibility to externally added immobilized protease. This cell-free system, consisting of components from an organism amenable to genetic analysis, will facilitate the study of the poorly understood mechanism of nuclear protein localization. The finding that ATP hydrolysis is required for nuclear import is the most direct evidence that nuclear localization is energy-dependent.