The kinetics of in vivo clearance of apolipoprotein (apo) A-I radioiodinated by the iodine monochloride (ICI) method of McFarlane [McFarlane, A.S. (1958) Efficient Trace-Labelling of Proteins with Iodine, Nature 182, 53] as modified by Bilheimer and co-workers [Bilheimer, D.W., Eisenberg, S., and Levy, R.I. (1972) The Metabolism of Very Low Density Lipoprotein Proteins. I. Preliminary in vitro and in vivo Observations, Biochim. Biophys. Acta 260, 212-221] and by using the IODO Beads Iodination Reagent were evaluated in rabbits. Both human apoA-I and rabbit HDL radioiodinated by the IODO Beads Iodination Reagent were cleared faster from plasma of rabbits than those radiolabeled by the ICI method. However, the different radiolabeling procedures in the ICI method, i.e., apoA-I radiolabeled either exogenously or in situ as a part of intact HDL, were not associated with a significant difference in the in vivo kinetics of apoA-I in rabbits if apoA-I was prepared by the guanidine HCI method and used fresh. 125I-ApoA-I subjected to delipidation and lyophilization was cleared only slightly faster from the plasma of rabbits than fresh 125I-apoA-I. We also found that apoA-I separated by the guanidine HCI method and used fresh was cleared faster from the plasma of rabbits when it was injected as free apoA-I without adding serum albumin or after in vitro incubation with rabbit HDL than when injected after reassociation with rabbit plasma. We conclude that the ICI method is a more appropriate radioiodination method for studying the in vivo kinetics of HDL than the IODO Beads Iodination Reagent and that the in vitro incubation conditions before injection are important factors that affect the in vivo kinetics of apo A-I.