A double-blind study of the effect of biotin supplementation (0.9 mg/day) of self-selected diets on plasma lipids and other plasma constituents was carried out in 40 men and women, age 30 to 60 years, for 71 days. Comparison of percent change from control levels showed significant treatment effects on more plasma constitutents than did comparison of the means of the actual levels. In an analysis of period of period changes, the largest differences were observed generally during the first two weeks after supplementation when small but statistically significant positive changes for biotin-treated men and women differed significantly from changes for the placebo-treated in total lipid, total phospholipid, and alpha + beta lipoprotein cholesterol. At the end of the study, these levels were at or below initial levels. Plasma biotin levels were elevated by biotin supplementation. There was a negative correlation between biotin levels and total plasma lipids. Responses os some lipid constituents of plasma were greater in volunteers who initially had elevated levels of lipids than in those who initially had normal levels of lipids. It is concluded that human requirements for biotin should be studied by new approaches and with modern techniques. Questions raised regarding the role bo biotin in the lipid metabolism of normal men and women should be investigated further.