Abstract <p>The knockout of either <i>At3g01500</i> or <i>At3g52720</i> gene encoding <i>Arabidopsis thaliana</i> βCA1 and αCA1 carbonic anhydrase (CA), respectively, led to a lower CA activity of the chloroplast stroma preparations from the knockout mutant plants (αCA1-KO and βCA1-KO) compared to such preparations from the wild-type (WT) plants. To identify the differences in the photosynthetic characteristics of mutant and WT plants, they were grown in low light (LL; 50-70&#xa0;µmol quanta∙m⁻²∙s⁻¹, natural conditions) and high light (HL; 400&#xa0;µmol quanta∙m⁻²∙s⁻¹, stressful conditions). The rate of CO₂ assimilation measured at 400&#xa0;µmol quanta∙m⁻²∙s⁻¹ in plants grown under LL was lower in αCA1-KO and βCA1-KO mutants compared to WT plants. The rate of photosynthetic electron transport was lower in αCA1-KO plants and higher in βCA1-KO plants than in WT plants; the content of CO₂ in chloroplasts was lower in βCA1-KO plants than in both WT and αCA1-KO plants, where it differed little. The value of the proton-motive force was higher in βCA1-KO plants and lower in αCA1-KO plants than in WT plants due to changes in ΔpH value. The obtained results suggest that βCA1 facilitates the intake of inorganic carbon into chloroplasts, while αCA1 ensures the conversion of bicarbonate into CO₂ in the chloroplast stroma for its use in the reaction catalyzed by Ribulose bisphosphate carboxylase/oxygenase (RuBisCO). In both αCA1-KO and βCA1-KO mutants, the expression levels of genes encoding other chloroplast CAs differed markedly from those in WT plants; the pattern of the changes in the genes expression depended on the light intensity during cultivation. The content of hydrogen peroxide in the leaves of both αCA1-KO and βCA1-KO mutants was higher in LL and lower in HL than in WT plants. The expression levels of stress marker genes changed similarly in both types of mutant plants. A&#xa0;possible involvement of the chloroplast stroma CAs in the transmission of stress signals in higher plants is discussed.</p>