Recently, p73 was identified as a structural and functional homolog of p53. The p73 protein activates the transcription of genes downstream of p53 and induces apoptosis when overexpressed in several cell lines, similar to the tumor suppressor p53. However, the extracellular stimuli and molecular mechanisms regulating p73 activity remain to be elucidated. In this paper, we present evidence that the naphthoquinone analog, 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (NA), is a novel apoptotic stimulus that induces p73beta expression. Treatment with NA induced the expression of p73beta mRNA and protein and its downstream genes, p21 and bax, in HeLa cells. Similar results were obtained in MCF7 cells (p53(+/+), p73(+/+)). In the MCF7 cells, p53 protein level was rather decreased by NA treatment. Overexpression of p73beta led to the apoptosis of HeLa cells and enhancement of NA-induced cell death. Expression of p73beta was mediated by E2F-1, which was activated via release from pRB after exposure of cells to NA. We additionally observed that overexpression of pRB inhibited NA-induced apoptosis. These results imply that p53-independent p73beta-dependent p21 expression is involved in NA-induced apoptosis of HeLa cells.