S U M M A R YProthymosin ␣ is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin ␣ appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin ␣ molecules, but not prothymosin ␣ fused to  -galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin ␣ shared domains with mobile proteins such as Ran, transportin, and karyopherin  , which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin ␣ . The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin ␣ is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus. T he function of prothymosin ␣ remains unclear despite many attempts to determine both its binding partners and its cellular location. Although the levels of the protein and its mRNA unquestionably increase in proliferating mammalian cells (Eschenfeldt and Berger 1986;Gómez-Márquez et al. 1989;Bustelo et al. 1991;Tsitsiloni et al. 1993), precisely what prothymosin ␣ does, where it does it, and what other macromolecules participate are questions with multiple unsatisfactory answers. The difficulties stem from three attributes: (a) Prothymosin ␣ is extremely acidic, with a net negative charge of ف 40 units (Eschenfeldt and Berger 1986;Goodall et al. 1986;Frangou-Lazaridis et al. 1988;Panneerselvam et al. 1988;Schmidt and Werner 1991). The large number of contiguous acidic amino acids in a protein of only ف 12 kD (Haritos et al. 1985), an unfolded conformation (Gast et al. 1995), and an abundance approaching that of histone cores (Palvimo and Linnala-Kankkunen 1990;Sburlati et al. 1990Sburlati et al. ,1993 make prothymosin ␣ an easy target for basic proteins in binding studies performed in vitro. The relevance of binding data is, accordingly, problematic. (b) Antibodies raised to prothymosin ␣ or peptides derived from it are invariably low in titer and broad in specificity. Such antibodies, particularly those directed against the N-terminus, crossreact with materials from crabs, insects, protozoans, fungi, and bacteria (Oates and Erdos 1989). However, the fully sequenced yeast genome does not code for prothymosin ␣ or related proteins, and homologous proteins in bacteria and homologous genes in a broad swath of the evolutionary spectrum up to and including reptiles h...