A novel carbonate microsensor, based on the ion‐selective ionophore N,N,‐dioctyl‐3a,12 a‐bis(4‐trifluoroacetylbenzoxy)‐5β‐cholan‐24‐amide, is presented. The sensor chemistry and filling electrolyte, used previously for macrosensors, was improved for use in microsensors, and a simple calibration procedure was designed. The sensor is highly selective for carbonate, having a similar selectivity as the macrosensor, and is so insensitive to Cl− interference that it can be used in seawater. The ability to measure accurate profiles with the carbonate sensor was verified in agar gels with artificial carbonate gradients. Several environmental applications are presented, including photosynthesis and calcification measurements in freshwater stromatolites (tufas) and foraminifera. Carbonate profiles in illuminated and darkened hypersaline microbial mats were qualitatively as expected and aligned with the oxygen and pH profiles. The dissolved inorganic carbon profiles calculated from local pH and carbonate values, however, did not follow the expected trends, both in the foraminifera and the hypersaline mat. Temporal and spatial heterogeneities make perfect alignment of pH and carbonate profiles, needed for DIC calculations, unrealistic. The calculation of dissolved inorganic carbon microprofiles from pH and carbonate microprofiles is not recommended. The microsensor is highly useful in studies on calcification and decalcification, where direct concentrations of carbonate and calcium ions are needed
