To assess the potential relationship between p53 and p16 proteins in the cellular response to stress, we have examined the levels of these proteins in a series of human tumor cell lines after treatment with either ionizing radiation or hyperthermia. We found that cells with abnormal radiation-induced G1 arrest (non-functional p53) had significantly higher constitutive levels of p16 than cells showing a normal G1 arrest (functional p53). Time-course experiments were done to test the effect of -irradiation on intracellular levels of p16. The pattern of changes in p16 response was similar in all cell lines studied, and p16 expression was not related to cellular sensitivity to radiation or to the level of p53 induction after treatment. We also provide evidence that short-term exposure to high temperature causes p53 accumulation. Hyperthermia-induced p53 accumulation was greatest in those cells exhibiting the highest radiation-induced p53 accumulation, suggesting a possible relationship between p53 induction after these 2 different stresses. p16 synthesis was also induced in different cell lines after heat treatment, and this response was independent of p53 functionality. When we compared the level of p16 expression with the extent of G0/G1 arrest induced by heat, a linear correlation was found, raising the possibility that p16 may be involved in the control of cell cycle progression in response to heat treatment.