High density lipoprotein 2 (HDL2) was incubated with phospholipid transfer protein (PLTP) or with hepatic lipase (H-TGL), and the incubation products were separated into a d < 1.22 g/ml and a d > 1.22 g/ml fractions. The d < 1.22 g/ml fraction produced by PLTP was larger, had lower apolipoprotein A-I and higher lipid and apolipoprotein A-II content than native HDL2. The d > 1.22 g/ml fraction represented 30% of the initial HDL2 protein and consisted of small, apolipoprotein A-I and phospholipid-rich particles, with a high sphingomyelin:phosphatidylcholine ratio. Incubation with H-TGL led to a d < 1.22 g/ml fraction which was comparable to native HDL2 regarding size and chemical composition. The d > 1.22 g/ml particles represented only 5% of the initial HDL2 protein and had slightly higher diameter and sphingomyelin:phosphatidylcholine ratio than those produced by PLTP. Enrichment of HDL2 with triglyceride prior to incubation increased the amount of protein released into the d > 1.22 g/ml fraction (20%) but had no effect on size and chemical composition of the particles. We conclude that PLTP and H-TGL promote the formation of small, pre-beta-like HDL particles from HDL2.