Lipoproteins, the major nutrient source for developing embryos in egg-laying species, are thought to be transported from the circulation of the hen to the yolk of growing oocytes. In order to fully understand the contribution of the different lipoprotein species to oocyte growth, yolk formation, and embryo development, we have started to elucidate the relationships between the high density lipoproteins (HDL) in serum with the hitherto uncharacterized yolk HDL fraction. Immunoblotting with antibodies against apolipoprotein (apo) A-I, the major protein moiety of circulating HDL, revealed, for the first time, significant amounts of this protein in yolk. Importantly, yolk apoA-I was an integral component of bona fide lipoprotein particles: i) the apoA-I-containing particles could be purified by ultracentrifugal flotation and immunoaffinity chromatography on immobilized anti-apoA-I IgG; ii) the particles resembled serum HDL in ultrastructural, chemical, and biochemical aspects; and iii) in particular, these particles contained another major apolipoprotein, apo II. To date, apo II has been assumed to be unique to the very low density lipoprotein (VLDL) and HDL fractions of laying hen serum. Its residence on yolk HDL particles, together with the other results, strongly implies that yolk HDL, at least to a large part, is derived from serum. This implication is supported by the presence of apoA-I in oocytic coated vesicles. However, an oocyte plasma membrane receptor for the transport of HDL could not be identified; furthermore, immunoelectron microscopy demonstrated that yolk HDL particles do not colocalize with VLDL, known to be endocytosed via a specific receptor. Thus, these studies have revealed that HDL particles are taken up into the oocyte from the serum of the laying hen, and are deposited into the yolk by a mechanism distinct from that involved in the uptake of other yolk lipoproteins.