The aim of this study was to determine the mechanisms responsible for the growth inhibitory effect of hyperthermia and verapamil in human colon cancer cell line HT-29. Apoptotic cell death was verified by flow cytometry analysis. The effect of treatment with hyperthermia and verapamil on the expression of apoptosis-associated proteins including Bcl-2, p53, bax, and c-Myc was studied by Western blot analysis. Changes in intracellular calcium homeostasis was analysed by fluorescence microscopy. The combination of 42 degrees C hyperthermia and verapamil caused a significant delay of human colon cancer cell proliferation as a result of apoptosis. Administration of these agents alone did not cause any cell inhibitory effect. Our experiments have shown that HT-29 cells constitutively express apoptosis-promoting proteins, such as Bax and c-Myc, while they fail to produce Bcl-2. Therefore, we hypothesize that HT-29 cells must have Bcl-2 independent pathways to protect cells against death-inducing signals. Also, apoptosis of HT-29 cells produced by hyperthermia in the presence of verapamil is a p53-independent process. Verapamil, when it did not act as a calcium channel blocker or inhibitor of release from intracellular storages under hyperthermic conditions, accelerated the increase of [Ca2+]i in HT-29 cells which resulted in programmed cell death (apoptosis).