Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m(2), but not at 150 J/m(2), induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m(2) UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m(2) UVB, but not at 150 J/m(2). However, with both 150 and 500 J/m(2) UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m(2) UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.