To permit CO2 measurements behind stomatal pores of plant leaves a potentiometric CO2-biosensor was built with a tip diameter of 2μm. The miniaturised sensor assembly consists of a H+ carrier-based pH-microelectrode concentrically arranged within a sheathing micropipette. The tip of the latter is filled with carbonate buffer, the pH of which quickly responds to CO2 concentration changes due to incorporated carbonic anhydrase. The CO2-microsensor shows a linear response to CO2 in the physiologically relevant concentration range of 50–800ppm (5.1–81.1Pa). The 90% response time varied from sensor to sensor between 18 and 63s (mean±S.E., 42±14s; n=6). Sensor calibration and leaf experiments were performed in an open-flow tube-like minicuvette, allowing tangential air flow along the leaf surface with controlled gas mixtures and flow rates of choice. At 800ppm external CO2 concentration, CO2 within the leaf was close to the external CO2 concentration when stomatal pores were wide open. However, the concentration dropped to 350ppm during stomatal closure due to CO2 consumption by photosynthesis demonstrating distinct CO2-sensing of internal leaf CO2. Following ‘light-off’ internal CO2 rapidly rose close to 700ppm, a response which was completely reversed by ‘light-on’. We conclude that this sensor is a suitable tool for CO2 monitoring in places too small to be accessible to conventional tools.