Peptide analogues of the class A amphipathic helixes from exchangeable apolipoproteins mimic apolipoprotein (apo) A-I in a number of ways, including the ability to activate the enzyme lecithin:cholesterol acyltransferase, to associate with high density lipoproteins (HDLs), and to form HDL-like particles in the presence of lipids. This study investigated the metabolic properties of several of these peptide analogues in the rat. Peptide analogues studied were 18A (referred to as L-18A to differentiate it from D- 18A, and which mimics apolipoprotein amphipathic helical domains in its charge distribution), 37pA (a dimer of two 18A monomers separated by a proline), 18R (with reversed charge distribution compared with 18A), and D- 18A (identical in amino acid sequence to 18A but synthesized from D-amino acids). Peptides were radiolabeled with 125I. In addition, metabolism of rat and human 125I-apo A-I and human 14C-apo A-I was studied; no significant differences in clearance of these preparations were seen. Clearance data were fitted to multiexponential equations to give half-times of clearance; biexponential equations consistently provided the best nonlinear least-squares curve fit. The order of relative lipid affinity determined in vitro was 37pA>apo A-I>D-18A=L-18A>18R. Half-times of clearance were in the same approximate rank order: 37pA, 6.9±3.3 hours (mean±SD); apo A-I, 6.9±1.8 hours; D-18A, 4.0±1.0 hours; L-18A, 4.6±1.6 hours; and 18R, 0.9±0.1 hour. Rats injected with L-18A had five times more radioactivity in the urine than did rats injected with D-18A. All urine radioactivity was present as free 125I in rats injected with L-18A or apo A-I but was present as intact peptide (with no free 125I) in rats injected with D-18A. The majority of radioactivity in L- and D-18A-injected rats was associated with the thyroid gland (in the case of L-18A), the liver, and the kidney. In summary, the rates of clearance of amphipathic helical peptides from the plasma compartment in rats decrease as the affinities of the peptides for lipoprotein surfaces increase. Stereoconformation did not affect the rate of clearance of peptide analogues. Although a significant proportion of radioactivity in L and D-18A-injected animals was associated with the kidney, excretion of intact peptides in the urine did not appear to be a major route of clearance.