The involvement of DNA damage in heat shock-induced cell death remains controversial. To investigate whether heat shock can induce DNA damage, we tested the induction of gammaH2AX foci formation, a sensitive indicator for DNA double strand breaks (DSBs), by heat shock treatment in several cell lines including HeLa, CHL, HepG2, and 293 cells, as well as human spermatozoa. Although heat shock treatment can decrease cell viability, no induction of gammaH2AX foci formation was observed in any of these cells. In addition, a p53-deficient cell line (U2OSE6tet24) and a flap endonuclease 1 (FEN1)-deficient cell line (FL-FEN1(-)) also did not show induction of gammaH2AX foci after heat shock treatment. Finally, it was found that 30min of pre-heat shock can inhibit gammaH2AX foci formation induced by an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which is known to induce gammaH2AX foci formation. On the other hand, heat shock after MNNG treatment did not affect the gammaH2AX foci formation induced by MNNG. Taken together, these data suggest that although heat shock might influence the gammaH2AX foci formation process, it does not induce DNA damage in the cells tested in this study.