Five patients received cholesterol-7alpha-(3)H intravenously during control periods. Specific activity of total serum cholesterol was determined serially during the 1st wk and weekly thereafter. 28-59 wk after the injection of the tracer, when no further radioactivity could be detected in serum cholesterol, 2 g of oral neomycin was given daily to four patients for the remainder of the experiment. Average total serum cholesterol concentrations were reduced by 20, 21, 26, and 29%, respectively, in these subjects. The fifth patient, given placebo, had no change in serum cholesterol. After a period of 12-26 wk of medication the intravenous injection of cholesterol-7alpha-(3)H was repeated, and while neomycin or placebo administration was continued, serum cholesterol specific activity was again determined serially during the 1st wk and weekly thereafter for 23-42 wk. The data were subjected to a two-compartment analysis. During the administration of neomycin, half-times of the cholesterol radio-activity decay curves were decreased in two patients and remained unchanged in two subjects. The size of the "intermediate" pool of cholesterol decreased in each patient during the administration of neomycin by 33, 36, 40, and 44%, respectively. The absolute decrease was much larger in each case than the concomitant reduction of serum cholesterol. There was no significant change in the data during the administration of placebo in one patient. The size of the "intermediate" pool can be calculated by compartmental analysis from the cholesterol decay curves. For the "slow" pool size and the other kinetic parameters only ranges of values can be deduced from the present experiment.