The in vitro metabolism of fructose and glucose and the glycerol release by adipose tissue of normal fed, fasted-refed, acutely and chronically diabetic rats were studied and the effects of insulin were investigated. Glycerol release by adipose tissue of fasted-refed rats exceeded that of normal fed and diabetic rats. In all four groups of animals that were studied, basal glycerol release was higher with fructose than with an equal concentration of glucose in the medium. Insulin inhibited glycerol release by adipose tissue in all groups of rats with fructose in the medium. In fed rats a barely significant insulin inhibition occurred also in the presence of glucose, and in fasted-refed rats it was independent of whether a hexose was present in the medium or not. The following effects of insulin on tissue of fasted-refed rats incubated in the absence of hexose were observed: Inhibition of glycerol release with a dose effect relationship between 10 and 250 µunits insulin per milliliter; partial inhibition of glycogen breakdown during incubation; diminished lactic and pyruvic acid release and a rise of the lactic to pyruvic acid ratio. These same effects were also exerted by the antilipolytic drug 5-methylpyrazole-3-carboxylic acid, which does not stimulate glucose uptake. At comparable rates of hexose uptake insulin decreased incorporation of hexose-14C into glyceride-glycerol and, conversely, increased fatty acid synthesis. These deviations of hexose-14C metabolism due to insulin could be explained as sequelae of the antilipolytic effect of insulin. A hypothesis is advanced according to which insulin might block the activity of lipoprotein lipase toward intracellular triglycerides while increasing its activity toward extracellular triglycerides.