This study was undertaken to measure and compare the rates at which digitonin-precipitable sterols (DPS) were synthesized in vivo in the major organs of five different animal species. These rates were assessed by measuring the velocity at which [3H]water was incorporated into DPS in the intact animal. The animals used were chosen to include species that carried most of their plasma cholesterol either predominantly in high (rat, hamster) or low (guinea pig) density lipoproteins (HDL and LDL, respectively) or more evenly distributed between the LDL and HDL fractions (monkey and rabbit). Whole animal sterol synthesis was much higher in the rat (16.1 mumol/hr) than in the other four species (2.9-4.6 mumol/hr) when normalized to a constant body weight of 100 g. This uniquely high rate of sterol synthesis could be attributed predominantly to an extremely high rate of incorporation of [3H]water into DPS by the liver of the rat. When expressed per g of tissue, the highest content of newly synthesized sterol in all species was found in tissues such as adrenal gland, ovary, and gastrointestinal tract. However, the content of [3H]DPS in the liver varied markedly from a high of 2279 nmol/hr per g in the rat to a low of only 109 nmol/hr per g in the guinea pig. Consequently, when expressed as a percentage of total body synthesis, the whole liver of the rat contained 51% of the [3H]DPS while this figure was much lower in the monkey (40%), hamster (27%), rabbit (18%), and guinea pig (16%). Thus, in all species except the rat, the major sites for sterol synthesis appeared to be the gastrointestinal tract, carcass (predominantly the muscle), and skin. In addition, even though the content of newly synthesized sterol per g of adrenal gland was higher than in nearly any other tissue in all of the species examined, it was further demonstrated that in the rat most of this [3H]DPS was derived from the blood (and, therefore, ultimately from the liver) whereas in the other species it was largely synthesized locally within the gland. Thus, these studies demonstrated that in many species the liver is quantitatively far less important as a site for sterol synthesis than previously believed and, as a correlate of this, most sterol utilized by extrahepatic tissues is largely synthesized locally within those tissues.