Adiantum capillus-veneris belongs to the Leptosporangiopsida, which are newly diversified fern species that grow in shadow beneath angiosperms (Schneider et al., 2004), and usually makes its habitat beneath the canopy, where the ambient light is weak and rich in far-red. The stomata are found only on the lower surface of leaves and lack subsidiary cells, and their guard cells contain densely arranged chloroplasts. Recently, we reported that the stomata of the fern A. capillus-veneris lacked a blue light-specific opening response but did open in response to red light (Doi et al., 2006). A lack of blue light-specific response has commonly been seen in other species of Leptosporangiopsida. We also showed that phy3 (neochrome 1; Suetsugu et al., 2005), a chimeric protein of phototropin and phytochrome, did not work as a photoreceptor for red light-induced stomatal opening in Adiantum. Therefore, the red light-induced stomatal opening in Adiantum could be explained by the photosynthetic activity and/or by the response via phytochrome. Here, we investigated the stomatal responses of Adiantum to light and CO₂ in the dark. We demonstrated that the guard cells are insensitive to CO₂ and that the guard cell chloroplasts are responsible for stomatal opening in response to light in this plant species.