Phospholipid transfer protein (PLTP) converts high density lipoproteins (HDL) to larger and smaller particles during incubation in vitro. The present study shows that these conversions are enhanced in triglyceride (TG)-enriched HDL. When pig HDL were enriched with TG by incubation with cholesteryl ester transfer protein (CETP) and very low density lipoproteins, the TG/apoA-I molar ratio increased from 0.7/1 to 11.4/1 and the diameter increased from 9.0 to 9.5 nm. When unmodified pig HDL were incubated with PLTP, the diameter increased from 9.0 to 9.5 nm. Incubation of the TG-enriched pig HDL with PLTP generated larger (13.0 nm) and smaller (7.5 nm) particles. The composition of the large particles was comparable to the non-incubated, TG-enriched HDL. The small particles contained only phospholipids and apolipoprotein (apo) A-I. Studies were also carried out with spherical, reconstituted HDL (rHDL) containing only cholesteryl esters in their core. When the rHDL were enriched with TG by incubation with Intralipid and CETP, the diameter increased from 9.0 to 9.2 nm and the TG/apoA-I molar ratio became 5.7/1. When unmodified rHDL were incubated with PLTP, the diameter of 15% of the particles increased to 11.0 nm, 18% decreased to 7.6 nm, and 67% were unchanged. When the TG-enriched rHDL were incubated with PLTP, the diameter of 62% of the particles increased to 11.4 nm, 26% decreased to 7.6 nm, and 12% did not change. Preincubation with Intralipid alone or CETP alone did not enrich the rHDL with TG or enhance the size changes. These results show that the TG content of HDL has a major influence on PLTP-mediated size changes.