The effects of 5, 10 and 20% dietary menhaden oil (MO) on the composition of heart lipid classes and fatty acids were studied. Male Sprague-Dawley rats were fed ad libitum 0, 5, 10 and 20% MO for 3 wk. The heart phosphoglyceride content and composition and cholesterol were unchanged by dietary MO. A nonlinear dose-response relationship was observed between dietary MO levels and fatty acid compositional changes. Cardiolipin, choline (PC), ethanolamine (PE) and serine/inositol (PS/PI) phosphoglycerides showed an incorporation of n-3 fatty acids, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3), between the control and 5% MO group, a plateau between the 5 and 10% MO groups and a further increase at the 20% MO level. The initial reduction in 20:4n-6 content remained unchanged as dietary MO increased except in PE where a further reduction was found at the 20% MO level. Dietary MO did not significantly change the 20:4n-6 content in neutral lipids. Linoleic acid content was most resistant to dietary MO removal. The level of 18:2n-6 was significantly lowered in heart PC when rats were fed 10% MO. No significant differences were found in PS/PI. In PE and NL significant differences occurred only when rats were fed 20% MO. The significant fatty acid modifications of heart lipid and PL found between the control and lowest level of dietary MO (5%) suggest that dietary fish oil supplementation in human diets may not be required for this effect.