Rat serum high density lipoproteins were divided into two fractions, HDL(2) (d 1.063-1.12) and HDL(3) (d 1.12-1.21). These fractions were compared on the basis of (a) the pattern of the apolipoprotein peptides obtained on polyacrylamide gel electrophoresis in 7 m urea, (b) the exchange of some of the peptides with those in very low density lipoproteins (VLDL), and (c) the incorporation by perfused rat liver of [(3)H]leucine into the peptides of the HDL(2) and HDL(3) secreted into the perfusate. Among the peptide bands of HDL(3), one is absent and another present only in trace amounts in HDL(2). After electrophoresis on polyacrylamide gel for 24 hr, a major peptide band of HDL(2) is split into three distinct areas, whereas it remains as a single area in HDL(3). Both HDL(2) and HDL(3) exchange prelabeled protein with VLDL. However, the exchange is much more limited in HDL(3), even though it contains most of the protein found in circulating rat HDL. Analysis of the individual peptides, separated by polyacrylamide gel electrophoresis after incubation with VLDL, reveals that in HDL(3) the exchange is limited to two peptides, whereas a third, although present in both subfractions of rat HDL, exchanges only when found in HDL(2). This peptide represents most of the exchange with VLDL. Perfused rat liver incorporates [(3)H]leucine into HDL of the perfusate, primarily into HDL(2). Most of the radioactivity is found in those peptides that do not take part in the exchange with VLDL. These data lead to the conclusions that there are functional and structural differences between HDL(2) and HDL(3) and that some of the peptides of HDL may be derived from exchange with, and breakdown of, VLDL. Others are secreted, at least in part, directly into the circulation by the liver.