Previously we have shown that heat shock cannot induce DNA damage in several mammalian cell lines, using gammaH2AX foci formation as an indicator for DNA damage. However, there have also been reports showing heat shock can induce DNA damage. In an effort to clarify why such differences exist among different studies, we examined the effects of five different heating methods, e.g., (1) heat block; (2) submerged in water bath; (3) half-submerged in water bath; (4) pre-heated medium; and (5) heating oven, on gammaH2AX foci formation in human amnion FL cells and Chinese hamster CHL cells. It was found that during a 24 h period, heating at 42 degrees C for 30 min showed no significant cytotoxicity, while 45 degrees C exhibited various degrees of cytotoxicity. The ability of these five methods to induce gammaH2AX foci was quite distinct, with only heat block inducing gammaH2AX foci at both 42 degrees C and 45 degrees C in both FL and CHL cells. The two cell types also responded differently to the different heating methods, with CHL being more sensitive to heat shock. Taken together, it is clear that certain heating methods can induce gammaH2AX foci formation; on the other hand, their effects differ in thermo-tolerant and non-tolerant cells. And the difference in gammaH2AX foci formation observed in different reports most likely is a consequence of the different heating methods used in the study. However, expression of gammaH2AX may not be used as a surrogate of cell killing.