Stomatal density, leaf conductance and water relations can be affected by an increase in the concentration of atmospheric CO2, and thus affect plant productivity. However, there is uncertainty about the effects of elevated CO2 on stomatal behaviour, water relations and plant productivity, owing to the lack of long-term experiments in representative natural ecosystems. In this work, variations in stomatal density and index, leaf water relations and plant biomass of semi-natural grassland communities were analysed under field conditions by comparing plants in three different experimental set-ups (natural CO2 springs, plastic tunnels and mini-FACE systems). Natural degassing vents continuously expose the surrounding vegetation to truly long-term elevated CO2 and can complement short-term manipulative experiments. Elevated CO2 concentration effects on stomata persist in the long term, though different species growing in the same environment show species-specific responses. The general decrease in stomatal conductance after exposure to elevated CO2 was not associated with clear changes in stomatal number on leaf surfaces. The hypothesis of long-term adaptive modifications to stomatal number and distribution of plants exposed to elevated CO2 was not supported by these experiments on grassland communities. Elastic cell wall properties were affected to some extent by elevated CO2. Above-ground biomass did not vary between CO2 treatments, leaf area index did not compensate for reduced stomatal conductance, and the root system had potentially greater soil exploration capacity. Considerable between-species variation in response to elevated CO2 may provide a mechanism for changing competitive interactions among plant species.