Abstract.Synthesis and secretion of triacylglycerol and cholesterol ester by cultured hepatocytes were highest in the presence of oleic, palmitic, stearic and linoleic acid, and lowest with eicosapentaenoic (EPA) and docosahexaenoic acid. Cellular formation and secretion of triacylglycerol and cholesterol ester were reduced by 50–80% in the presence of EPA in comparison with oleic acid. Reduced formation of triacylgycerol and cholesterol ester was also observed when EPA was given together with oleic acid. EPA caused higher incorporation of (3H)water into phospholipid and lower incorporation into triacylglycerol and cholesterol ester as compared with oleic acid. Rat liver microsomes incorporated EPA‐CoA into triacylglycerol and cholesterol ester to a lesser extent than oleoyl‐CoA. Decreased formation of triacylglycerol and cholesterol ester was also observed when EPA‐CoA was given together with oleoyl‐CoA, whereas palmitoyl‐CoA, stearoyl‐CoA, linolenoyl‐CoA and arachidonoyl‐CoA had no inhibitory effects. In conclusion, inhibition of acyl‐CoA: 1,2‐diacylglycerol acyltransferase and acyl‐CoA: cholesterol acyltransferase by EPA may be important for decreased hepatic synthesis and secretion of triacylglycero