Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.