T he effect of feeding diets containing 75% glucose or fructose on liver triglyceride formation in the rat was studied by both in vivo and in vitro techniques. The results were compared with those from control rats fed laboratory chow.Both high-sugar diets increased the capacity for triglyceride formation from sn-glycerol-3-P by rat liver homogenates and correspondingly increased incorporation of [1,3-¹⁴C]glycerol into hepatic triglyceride by the intact animal.These independent measures of hepatic triglyceride production changed with a similar time-course characteristic for each diet. The 75% fructose diet produced a greater increase in both determinations, reaching a maximum after 11 days.Despite the increase in hepatic triglyceride formation by both high-sugar diets, only the 75% fructose diet resulted in a consistent and sustained increase in serum triglyceride. This results most probably from differences in the fractional rate of serum triglyceride removal between the two groups.When serum triglyceride removal was inhibited by administration of Triton WR-1339, both high-sugar diets increased incorporation of [1,3-¹⁴C]glycerol in serum triglyceride in vivo and increased serum triglyceride level above that in control rats.