The in vivo metabolism of esterified cholesterol in plasma HDL has been studied in rabbits injected with preparations of HDL which had been labeled with 3H in the esterified and free cholesterol moieties. These labeled HDL preparations had been isolated either from the serum of donor rabbits which had been previously injected with 3H-mevalonic acid or from rabbit serum which had been incubated in vitro at 37 degrees with 3H-cholesterol. In terms of subsequent in vivo metabolism, there were no significant differences between the in vivo labeled and in vitro labeled HDL preparations. It was also found that the presence of free 3H-cholesterol in the injected HDL had very little effect on the recipient esterified 3H-cholesterol results. The removal of esterified 3H-cholesterol from the recipient HDL fraction was biphasic, with the initial phase largely reflecting a transfer into the plasma VLDL and LDL fractions rather than a removal from the plasma compartment. In fact, the initial rate of removal from HDL was very much influenced by the distribution of esterified cholesterol mass in the different lipoprotein fractions; the greater the proportion in VLDL and LDL, the more rapid was the initial rate of removal from HDL. A transfer of esterified cholesterol from HDL to VLDL and LDL was estimated to be of the order of 150 to 200 mumol/L of plasma per hour, a value much greater than the reported rate of production of esterified cholesterol in rabbit plasma. The implication that such transfers might therefore have been partially reversible was confirmed in in vitro incubations of labeled HDL and unlabeled rabbit serum.