Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors.