Background: Essential fatty acid (EFA) deficiency during cholestasis is mainly due to malabsorption of dietary EFA (23). Theoretically, dietary phospholipids (PL) may have a higher bioavailability than dietary triglycerides (TG) during cholestasis. We developed murine models for EFA deficiency with and without extrahepatic cholestasis, and compared the efficacy of oral supplementation of EFA as PL or as TG. Methods: EFA deficiency was induced in mice by feeding a high-fat EFA-deficient (EFAD) diet. After three weeks on this diet, bile duct ligation (BDL) was performed in a subgroup of mice to establish extrahepatic cholestasis. Cholestatic and non-cholestatic EFA-deficient mice continued on the EFAD diet (controls), or were supplemented for three weeks with EFA-rich TG or EFA-rich PL. Fatty acid composition was determined in plasma, erythrocytes, liver and brain. Results: After four weeks of EFAD diet, induction of EFA deficiency was confirmed by a six-fold increased triene/tetraene ratio (T/T-ratio) in erythrocytes of non-cholestatic and cholestatic mice (p<0.001). EFA-rich TG and EFA-rich PL were equally effective in preventing further increase of the erythrocyte T/T-ratio, which was observed in cholestatic and non-cholestatic non-supplemented mice (12- and 16-fold the initial value, respectively). In cholestatic mice, EFA-rich PL was superior to EFA-rich TG in decreasing T/T-ratios of liver triglycerides and phospholipids (each p<0.05), and in increasing brain phospholipid concentrations of the long-chain polyunsaturated fatty acids (LCPUFA) docosahexaenoic acid and arachidonic acid (each p<0.05). Conclusions: Oral EFA supplementation in the form of PL is more effective than in the form of TG in increasing LCPUFA concentrations in liver and brain of cholestatic EFA-deficient mice.