The effect of dietary CLA and n-3 PUFA on hepatic TAG accumulation, histopathology, and FA incorporation in lipid classes by laying chickens was investigated. One hundred twenty 30-wk-old single comb white leghorn laying hens were distributed randomly to four treatments (3 replications of 10 birds) and were fed diets containing CLA and animal fat (Diet I), 18:3n-3 (Diet II), or long-chain n-3 FA (Diet III). A sunflower oil (n-6 FA)-based diet vvas the control. Feeding Diet I resulted in an increase in hepatic total lipids (P < 0.05). The liver TAG content was 32.2, 18.9, 29.4, and 18.7 mg/g for hens fed Diet I, Diet II, Diet III, and the control diet, respectively (P< 0.05). The serum TAG was lowest in birds fed Diet II (P < 0.05). Diet I resulted in an increase in the total number of fat vacuoles and lipid infiltration in hepatocytes (P < 0.05). The number of cells with 75% or higher lipid vacuolation was observed only in birds fed Diet I. Feeding diets containing CLA resulted in an increase in the content of the c9,t11 CLA isomer in liver TAG and PC (P < 0.05). No difference was observed in the CLA concentration of hepatic PE fractions. The content of DHA (22:6n-3) was higher in the TAG, PC, and PE of hens fed Diet II and Diet III than Diet I and the control (P < 0.05). Feeding CLA resulted in an increase in total saturated FA in the TAG and PC fractions (P < 0.05). Long-term feeding of CLA in laying birds leads to an increase in liver TAG and may predispose birds to fatty liver hemorrhagic syndrome.