Six adult pedigreed dogs were studied as long as 3 yr in order to determine the effects of cholesterol feeding and of bile diversion on absorption, synthesis, and storage of cholesterol. These measurements were based on cholesterol balance and isotopic kinetic studies. In the six dogs fed a 'cholesterol free' diet with their enterohepatic circulations undisturbed, the rate of cholesterol synthesis ranged from 225 to 508 mg/day. In two dogs studied subsequently on cholesterol containing diets, absorption of cholesterol averaged 81% on a dietary intake of 0.5 g/day; on high cholesterol intakes the rate of absorption dropped to 43-51% of daily intake, but the absolute amounts absorbed were increased. Feeding of cholesterol resulted in acceleration of bile acid formation and excretion, as well as nearly total inhibition of cholesterol synthesis. These two compensatory mechanisms were sufficient to maintain zero balance of cholesterol in the face of a high cholesterol intake. Plasma cholesterol concentrations in the two dogs increased by 37 and 44%. In two other dogs bile was completely diverted into the urinary system for nearly 2 yr. When these dogs were studied on a cholesterol free diet, the sum of acidic steroids excreted daily in urine plus neutral steroids excreted in feces was seven times as high as before the operation. The increased output of fecal neutral steroids could be the result of transfer of plasma cholesterol across the gut wall or due to increased synthesis in the gut. Plasma cholesterol levels were reduced in these two dogs by 20 and 27%, and triglycerides decreased by 36 and 43%. Accumulation of cholesterol in body pools in the cholesterol fed dogs appeared to have been prevented, according to antemortem measurements: increased absorption of dietary cholesterol was exactly balanced by suppression of cholesterol synthesis and enhanced bile acid excretion. In the bile shunted animals, depletion of tissue stores of cholesterol could not be predicted by antemortem measurements.