The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300?µM (37°C), heat shock (44°C per 20?min) alone or a combination of the two, then incubated at 37°C for 6?h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca 2+ concentration ([Ca 2+ ]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca 2+ ]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca 2+ ]i, the activation of the mitochondria–caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP. [ABSTRACT FROM AUTHOR]