Background: Photosynthetic cyanobacteria have been studied as potential host organisms for direct solar‑driven production of different carbon‑based chemicals from CO2 and water, as part of the development of sustainable future biotechnological applications. The engineering approaches, however, are still limited by the lack of comprehensive information on most optimal expression strategies and validated species‑specific genetic elements which are essen‑tial for increasing the intricacy, predictability and efficiency of the systems. This study focused on the systematic evalu‑ation of the key translational control elements, ribosome binding sites (RBS), in the cyanobacterial host Synechocystissp. PCC 6803, with the objective of expanding the palette of tools for more rigorous engineering approaches.Results: An expression system was established for the comparison of 13 selected RBS sequences in Synechocystis, using several alternative reporter proteins (sYFP2, codon‑optimized GFPmut3 and ethylene forming enzyme) as quan‑titative indicators of the relative translation efficiencies. The set‑up was shown to yield highly reproducible expression patterns in independent analytical series with low variation between biological replicates, thus allowing statistical comparison of the activities of the different RBSs in vivo. While the RBSs covered a relatively broad overall expres‑sion level range, the downstream gene sequence was demonstrated in a rigorous manner to have a clear impact on the resulting translational profiles. This was expected to reflect interfering sequence‑specific mRNA‑level interac‑tion between the RBS and the coding region, yet correlation between potential secondary structure formation and observed translation levels could not be resolved with existing in silico prediction tools.Conclusions: The study expands our current understanding on the potential and limitations associated with the regulation of protein expression at translational level in engineered cyanobacteria. The acquired information can be used for selecting appropriate RBSs for optimizing over‑expression constructs or multicistronic pathways in Synecho-cystis, while underlining the complications in predicting the activity due to gene‑specific interactions which may reduce the translational efficiency for a given RBS‑gene combination. Ultimately, the findings emphasize the need for additional characterized insulator sequence elements to decouple the interaction between the RBS and the coding region for future engineering approaches.