Fluorescent protein that detects caspase-3 activation was used for the time-lapse observation of CTL-target cell interaction. In the target cells transfected with SCAT3.1 (caspase-3-sensitive fusion protein) complementary DNA, caspase-3 activation can be detected significantly earlier than the commonly used annexin-V binding that detects membrane change in apoptotic cells. Moreover, during the cytolytic interaction between OE4 CTL and W3 tumor target cells, detachment of CTL from the target cells occurred prior to caspase-3 activation and death of the target cells, indicating very early sensing of apoptotic target cells by CTL. This early detachment of OE4 CTL from W3 target cells was inhibited by the expression of CD80 co-stimulatory molecule on the target cells. Taken together, time-lapse observation of cellular interaction with functional probe, SCAT3.1 provides new kinetic information and demonstrates that co-stimulatory molecules regulate the kinetics of CTL-target cell interaction.