Hybrid cells from the cross Nicotiana suaveolens x N. tabacum expressed hybrid lethality at 28 degrees C in a thin layer cell culture system. Features characteristic of apoptosis, such as DNA fragmentation, chromatin condensation and nuclear fragmentation, were detected during expression of hybrid lethality. Actinomycin D (ActD) or cycloheximide (CHX) added to the medium suppressed apoptotic cell death during hybrid lethality. This indicates that hybrid lethality requires de novo transcription and translation, and is thus under genetic control. To estimate the time course of apoptotic cell death during the expression of hybrid lethality, we determined when factors controlling hybrid lethality were expressed by observing the point of no return. When cells were exposed to 28 degrees C for 2 h or less in inhibitor-free medium before addition of ActD or CHX, the percentage of dead cells did not increase. However, when cells were exposed to 28 degrees C for 4 h before the addition of inhibitor, the percentage of dead cells increased. When cells were exposed to 28 degrees C for 3 h before the addition of inhibitor, the percentage of dead cells varied from experiment to experiment. These data indicate that the factors controlling hybrid lethality are expressed 3 h after induction of hybrid lethality. In addition, we found a time difference between the expression of cell death and nuclear fragmentation. This suggests that the factor controlling cell death is different from the one controlling nuclear fragmentation.