An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco sus-pension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei, evi-denced by DNA electrophoresis and fluorescence microscopy observations. Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still ex-hibited the apoptosis-inducing activity, but the supernatant had no effect, which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble. The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO, the specific inhibitor of Caspase-3, implying involve-ment of a Caspase-3-like protease during the process. Furthermore, extensive apoptosis in nuclei was in-duced by cytochrome b6/f on the thylakoid membrane, indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway. An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco sus-pension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei, evi-denced by DNA electrophoresis and fluorescence microscopy observations. Further study showed that chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still ex-hibited the apoptosis-inducing activity, but the supernatant had no effect, which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble. apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO, the specific inhibitor of Caspase-3, implying involve-ment of a Caspase-3-like protease during the process. b6 / f on the thylakoid membrane, indicating that this important cytochrome complex may have an important role in the chloroplast-related apo ptotic pathway.