A novel mode of tumor suppressor protein p53 regulation, mediated by recruitment of the linker histone H1 to the promoters of p53 target genes leading to specific repression of p53-dependent transcription, has recently been uncovered. Yet, how this repression could be relieved is not clear. Previously, a histone-binding nuclear protein prothymosin alpha (ProTa) was shown to trigger a p53 response. The histone-binding region of ProTa was found to be essential for this effect, allowing for the possibility that ProTa stimulates p53-dependent transcription by dissociating the p53-histone H1 repressive complex. In support of this model, here we show that ProTa interacts with the same C-terminal domain of histone H1 as p53 does and, therefore, ProTa and p53 could compete for binding to histone H1. Furthermore, ProTa, when competent for histone H1 binding, is able to liberate p53 from the histone H1-p53 complex in vitro. In vivo, stimulation of p53-dependent transcription by ProTa correlates with the ability of ProTa to interact with histone H1. Ectopic expression of histone H1 or its C-terminal ProTa-binding domain specifically suppresses the stimulating effect of ProTa on transcription of the p53-responsive reporter gene in cultured cells. These results are consistent with the model that ProTa may enhance p53 transcription activity by displacement of histone H1 from p53-H1 repressive complex.