Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the interconversion of CO2 and HCO3  − and is a major protein constituent of the C3 higher plant chloroplast where it is presumed to play a role in photosynthetic carbon assimilation. In this study, we have used both RNA antisense and gene knockout lines to specifically reduce the activity of the chloroplast βCA1 polypeptide (At3g01500) in the model plant Arabidopsis (Arabidopsis thaliana). Although able to germinate, seedling establishment of transgenic plants is significantly reduced relative to wild-type plants when grown at ambient levels of CO2. Growth at elevated (1,500 μL L−1) CO2 or on plates supplemented with sucrose restores seedling establishment rates to wild-type levels. Seed from wild-type and transgenic plants exhibited no significant differences in seed protein, lipid content, or reserve mobilization during seedling growth. βCA1-deficient seedlings do, however, exhibit reduced capacity for light-dependent 14CO2 assimilation prior to the development of true leaves. The small number of surviving seedlings able to grow and develop are phenotypically similar to wild-type plants, even when subsequently grown at subambient levels of CO2. Microarray analysis of mature leaves of βCA1-deficient plants shows some differences in transcript abundance, particularly with genes involved in ethylene signaling and response. The data suggest that reduced levels of seedling establishment by βCA1-deficient plants could be the result of poor cotyledon photosynthetic performance at the onset of phototrophic growth and prior to the development of true leaves.