Remodelling of plasma high density lipoproteins (HDL) promotes the dissociation of lipid-free apolipoprotein (apo)A-I from the particles. In the present study, we have investigated the formation of new HDL particles from lipid-free apoA-I in a process dependent on the presence of nonesterified fatty acids (NEFA) and other lipoprotein fractions (as donors of lipid). Incubations were carried out that included lipid-free apoA-I, VLDL, and lipoprotein lipase (LPL) or lipid-free apoA-I, either VLDL or LDL, and sodium oleate. Any new HDL particles that were formed were separated from lipid-free apoA-I in the ultracentrifuge. When any one of the ingredients in the incubation was absent, the apoA-I remained lipid-free; however, when all the ingredients were present, a significant proportion of the apoA-I was recovered in the HDL density fraction. This coincided with the formation of at least three HDL-sized subpopulations; one of the subpopulations was considerably smaller than HDL3c and had pre-beta 1 mobility while two were in the size range of human HDL2b and HDL3c and had pre-beta 2 electrophoretic mobility. The new HDL were predominantly discoidal in shape and their major constituents were apoA-I, phospholipid, and unesterified cholesterol. In conclusion, these results show that lipid-free apoA-I can form new HDL particles in the presence of NEFA and other lipoprotein fractions. The formation of pre-beta 1 HDL from lipid-free apoA-I indicates that this process is potentially of great importance in terms of generating plasma acceptors of cell cholesterol in reverse cholesterol transport.