Hsp72 is the major heat shock-inducible protein capable of protecting cells from a variety of stresses. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines. It is commonly assumed that in tumor cells the expression of Hsp72 at elevated levels is the consequence of oncogenic transformation. In the present study we addressed an alternative possibility that Hsp72 plays an active role in the process of oncogenic transformation. We report here that when Hsp72 was expressed in the Rat-1 fibroblasts either constitutively or from an adenovirus-based construct, cells become oncogenically transformed by the following criteria: loss of contact inhibition and formation of foci characteristic for oncogenically transformed cells; acquisition of the ability to grow in an anchorage-independent manner and to form colonies in soft agar; generation of tumors upon injection into mice. Furthermore, we also report that turning off the Hsp72 expression led to the reversal of the transformed phenotype. We also show that oncogenic potential of Hsp72 is confined in its peptide binding domain since the expression of this domain alone was sufficient for oncogenic transformation of Rat-1 cells.