Photoinhibition has been often evaluated with leaf discs floated on water or placed on wet papers to prevent desiccation. Under these conditions, there is a possibility that CO2 diffusion is blocked by water, which may lead to reduction in photosynthetic CO2 assimilation. Using Chenopodium album L. grown at two irradiances, photosynthesis, quantum yield of Photosystem II (ΔF/Fm′), non-photochemical quenching (qN), and photoinhibition were compared between detached leaves and leaf discs. In low-light-grown plants, photoinhibition was greater in leaf discs than in detached leaves, while in high-light-grown plants, there was little difference. Leaf discs showed lower rates of photosynthesis and ΔF/Fm′, and higher qN. The ΔF/Fm′ in leaf discs increased when leaf discs were exposed to high concentration of CO2, suggesting that CO2 diffusion to chloroplasts was limited in leaf discs floated on water.