ABSTRACT The biochemical and physicochemical properties of myofibrils and actomyosin (AM) from frozen stored fillets of flounder were investigated. Solubility of proteins increased after freezing and decreased during frozen storage. No significant (P < 0.05) changes were detected in both Mg2+ (Ca2+) and Mg2+ Ethylene glycol tetraacetic acid (EGTA) Adenosinetriphosphatase (ATPase) activities of myofibrils due to freezing and frozen storage. Freezing produced a significant (P < 0.05) increase in Mg2+ Ca2+ and Mg2+(EGTA) and Mg2+ATPase activities of AM. The (Ca2+) ATPase activity remained unchanged. All the ATPases activities of AM decreased during frozen storage. The changes in the enzymatic activities of AM during storage were accompanied by a decrease in the reduced viscosity of the protein. No signals of proteolysis were detected in the profiles of SDS-Polyacrilamyde Gel Electrophoresis 10% gels of both myofibrils and AM. A significant decrease (P < 0.05) in the relative percentage of myosin and myosin/actin ratio was observed at the end of storage. These results suggest that denaturation–aggregation of myosin occurs at the end of storage in frozen stored fillets. PRACTICAL APPLICATIONS Flounder (Paralichthys patagonicus) is an important species of fish in South American waters. In recent years, catches in Argentina greatly increased. The fishing is commonly called “fine fishing” because of the high quality of the meat and its high price in the market. It is processed and exported as frozen interfoliated fillets and consumed as fresh fish in the internal market. Only a few reports on changes in biochemical and physicochemical properties of both AM and myofibrils from iced flounder are available. However, there is a lack of information on the behavior of myofibrillar proteins from flounder (P. patagonicus) during freezing and frozen storage. This information could be very important to establish technological actions to obtain optimal quality in frozen flounder.