The purpose of the current study was to determine the effects of dietary docosahexaenoic acid (DHA) on fatty acid deposition in egg yolk and various tissues of laying Leghorn hens, and on the expression of hepatic lipogenesis-related transcription factors. There were three treatments with 10 Leghorn hens per treatment. Diets were based on maize-soybean meal (ME: 12 MJ/kg; CP: 171 g/kg; Ca: 34 g/kg) supplemented with 0 (plus 20 g butter/kg), 5 g (plus 15 g butter/kg) or 20 g algal DHA oil/kg. The egg production of the birds was not affected by dietary DHA oil (P>0·05). The DHA content in egg yolks of the laying hens increased significantly (P<0·01) with the dietary supplementation of DHA. The DHA content of the total fatty acids in the egg yolk of laying hens was 4±16, 11±43 and 20±90 g/kg for 0, 5 and 20 g/kg DHA treatments, respectively for the first week, and 6±16, 18±49 and 31±86 g/kg for the second week. Therefore, algal DHA oil can be utilized by laying Leghorn hens to enhance egg yolk DHA content and produce high DHA eggs. The concentrations of triacylglycerol (TG) and cholesterol in plasma of laying Leghorn hens were not affected by dietary DHA treatments (P>0·005). The DHA content in plasma and livers of laying hens was increased with the addition of DHA in the diet (P<0·05). The DHA content in the skeletal muscle of Leghorn hens was also increased with the increase in dietary DHA. The data indicate that dietary DHA oil can be incorporated into plasma, egg yolks and various tissues. The mRNA concentrations of the lipogenesis-related transcription factors, sterol regulatory element binding protein 1 (SREBP1) and SREBP2, in the livers of laying Leghorn hens were not affected by the supplement of algal DHA oil for 2 weeks, suggesting that the expressions of these transcription factors are tightly controlled and not sensitive to DHA treatments in laying hens.