There is currently significant interest in engineering the two-celled C4 photosynthesis pathway into crops such as rice in order to increase yield. This will require alterations to the biochemistry of photosynthesis in both mesophyll (M) and bundle-sheath (BS) cells, but also alterations to leaf anatomy. For example, the BS of C4 species is enlarged compared with that in C3 species. Because cell and nucleus size are often correlated, this study investigated whether nuclear endoreduplication is associated with increased differentiation and expansion of BS cells. Nuclei in the BS of C4Cleome gynandrawere tagged with green fluorescent protein. Confocal laser-scanning microscopy and flow cytometry of isolated nuclei were used to quantify size and DNA content in BS cells. The results showed a significant endoreduplication in BS cells ofC. gynandrabut not in additional C4 lineages from both the monocotyledonous and dicotyledenous plants. Furthermore, in the C3 speciesArabidopsis thaliana, BS cells undergo endoreduplication. Due to this significant endoreduplication in the small BS cells of C3A. thaliana, it was concluded that endoreduplication of BS nuclei in C4 plants is not linked to expansion and differentiation of BS cells, and therefore that alternative strategies to increase this compartment need to be sought in order to engineer C4 traits into C3 crops such as rice.