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Stomatal functioning regulates the fluxes of CO 2 and water vapor between vegetation and atmosphere and thereby influences plant adaptation to their habitats. Stomatal traits are controlled by external environmental and internal cellular signaling. The objective of this study was to quantify the effects of CO 2 enrichment (CE) on stomatal density (SD)-related properties, guard cell length (GCL) and carbon isotope ratio (δ 13 C) of a range of Arabidopsis thaliana ecotypes originating from a wide altitudinal range [50-1260 m above sea level (asl)], and grown at 400 and 800 ppm [CO 2 ], and thereby elucidate the possible adaptation and acclimation responses controlling stomatal traits and water use efficiency (WUE). There was a highly significant variation among ecotypes in the magnitude and direction of response of stomatal traits namely, SD and stomatal index (SI) and GCL, and δ 13 C to CE, which represented a short-term acclimation response. A majority of ecotypes showed increased SD and SI with CE with the response not depending on the altitude of origin. Significant ecotypic variation was shown in all stomatal traits and δ 13 C at each [CO 2 ]. At 400 ppm, means of SD, SI and GCL for broad altitudinal ranges, i.e. low (<100 m), mid (100-400 m) and high (>400 m), increased with increasing altitude, which represented an adaptation response to decreased availability of CO 2 with altitude. δ 13 C was negatively correlated to SD and SI at 800 ppm but not at 400 ppm. Our results highlight the diversity in the response of key stomatal characters to CE and altitude within the germplasm of A. thaliana and the need to consider this diversity when using A. thaliana as a model plant., (© 2016 Scandinavian Plant Physiology Society.)