Our laboratory previously showed that high concentrations of supplemental defatted microalgae (DMA, 10–15%) or flaxseed oil (FO,5%) altered fatty acid profiles of egg yolk and tissues of layers. This experiment was to determine if and how lower concentrations of these supplementations affected fatty acid composition of egg yolk, liver, adipose tissue, and plasma of layers. A total of 60 Shaver leghorn layer hens (individually caged, 20 wk old) were allotted into 6 groups (n = 10) and fed a corn soybean meal-basal diet containing 0, 3, or 5% DMA (Nannochloropsis oceanica, Cellana, Kailua-Kona, HI) and 0 (1.5% corn oil) or 1.5% FO (Dyets, Bethlehem, PA) for 6 wk. Data were analyzed by 2-way (2 by 3 factorial) ANOVA with or without time-repeated measurements using PC-SAS (Version 9.1, SAS Institute, Inc., Cary, NC).Although BW, ADFI, egg production rate, egg weight, or egg albumen, yolk, and shell were not affected by any of the dietary treatments at various time points, egg yolk color was changed (P < 0.05) from 7 to 13 (Roche color fan) with increasing concentrations of DMA. There was no diet effect on plasma concentrations of triglyceride, cholesterol, or uric acid. While concentrations of n-3 fatty acids in egg yolk and plasma were elevated by FO up to 9-fold (P < 0.05) starting at wk 1 or 2, such improvements by DMA were only 39 to 83% (P < 0.05). Although DMA also improved (P < 0.05) n-6/n-3 fatty acid ratios in egg yolk and plasma from 13 to 23 to 7–13, this effect was seen only in hens fed diets without FO. In the presence of 1.5% FO, only 5% DMA produced 8–9% increase (P < 0.05) in n-3 fatty acid concentration of egg yolk at wk 2 and 3. Fatty acid profiles of liver and adipose tissue (collected at wk 6) displayed responses to supplemental DMA and FO similar to those of egg yolk or plasma. In conclusion, supplemental 2.5 or 5% DMA caused moderate enrichments of n-3 fatty acids and decreases of n6/n3 fatty acid ratios of egg yolk and tissues in hens fed diets containing corn oil. The DMA effects on these measures were very limited in hens fed diets containing FO. Supported in part by a USDA/DOE Biomass R&D Initiative Grant and a Hatch Grant of Cornell University.