Abstract: Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) catalyzes the irreversible β-carboxylation of Phosphoenolpyruvate (PEP) to yield Oxaloacetate (OAA) and inorganic Phosphate (Pi). PEPC contributes to photosynthetic and anaplerotic CO2 fixation in higher plants and bacteria. The aim of this study was to determine the physicochemical properties of cyanobacterial PEPCs and to develop 3-dimensional models of selected enzymes. The biocomputational analyses were performed in silico using web-based software and servers. The alignment of cyanobacterial enzymes and secondary structure analysis revealed that there are conserved amino acid substitutions and polymorphisms between PEPCs from marine and fresh water organisms. Furthermore, some marine subgroups seem to possess unique amino acid stretches that may modulate various aspects of catalysis and regulation. Phosphoenolpyruvate carboxylase from Synechococcus PCC 7002, a marine organism, most closely resembles PEPC from fresh water organisms; for this reason, the enzyme was chosen for homology modeling alongside PEPCs from the fresh water strain Anabaena variabilis and the marine cyanobacterium Synechococcus RS 9917. Amino acids and domains that can distinguish between the fresh water and marine PEPCs were identified. Secondary structure analysis and homology modeling suggested that cyanobacterial PEPCs are primarily alpha helical with additional β-sheets flanking the characteristic central β-barrel. The physicochemical characteristics and the 3D models provide a framework for the purification and characterization of cyanobacterial PEPCs.