Mutants of human prothymosin α with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin α-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin α by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin α 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 α through an unknown mechanism, without influencing the nuclear import of the protein.