The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach and Arabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized the Arabidopsis At-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. The At-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage in Arabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants.