BACKGROUND: Fish by-products are not considered as valuable raw materials even if they usually contain valuable components such as lipids. Fish lipids are well known for their nutritional potential and health effects but their extraction remains problematic due to the use of organic solvents. Enzymatic hydrolysis such as the proteolysis of tissues can lead to lipid extraction. RESULTS: Hydrolysis of sardine heads by Protamex was studied (temperature, hydrolysis time and enzyme-substrate ratio) using response surface methodology in order to obtain the highest release of lipids and particularly phospholipids. No relation between the degree of hydrolysis and lipid recovery were depicted; however, optimum conditions for both the release of lipids and phospholipids were found to be similar (29 min, 31 degrees C with 2.6 g kg(-1) enzyme). Under these hydrolysis conditions, rich lipid and phospholipid fractions (oily and aqueous fractions) can be recovered when time, temperature and enzyme consumption are minimized. Analytical data have revealed that they contain high-quality lipids, especially omega 3 fatty acid. CONCLUSION: This study demonstrated that proteolysis can be used for high lipid recovery from little-exploited biomass like fish heads without requiring solvent or thermal treatment. Resulting phospholipids, fatty acids and peptides could be utilized for nutritional or feed purposes.