Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was the most tolerant to ethanol. Nevertheless, to implement a production process using recombinant strains, the bioethanol produced will be required to be continuously extracted from the culture media via a membrane-based technological process for example to prevent detrimental effects on the biomass. The results presented here are of significance in defining the maximum threshold for bulk ethanol concentration in production media.