The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.