The hepatic lipase acting on triglyceride-rich high-density lipoprotein₂ (HDL₂) induces the formation of preβ₁-HDL, leaving a residual alpha-migrating HDL particle that was named “remnant-HDL₂” (Barrans, A., Collet, X., Barbaras, R., Jaspard, B., Manent, J., Vieu, C., Chap, H., and Perret, B. (1994) J. Biol. Chem. 269, 11572−11577.]. In this study, these two product particles generated by hepatic lipase were isolated by density gradient ultracentrifugation. Particles were first characterized in terms of chemical composition, density, and mass. The preβ₁-HDL obtained in vitro contain one to two molecules of apoA-I, associated with phospholipids, and free and esterified cholesterol. When compared to triglyceride-rich HDL₂, remnant-HDL₂ have lost on average one molecule of apoA-I, 60% of triacylglycerols, and 15% of phospholipids. The estimated composition is concordant with the hypothesis of the splitting of a substrate particle into one preβ₁-HDL and one remnant-HDL₂. Spectroscopic studies were carried out to monitor changes in lipid fluidity upon lipolysis. The fluorescence anisotropy was measured using (1,6)-diphenyl-hexa-(1,3,5)-triene as a probe, and the degree of order was calculated from electron spin resonance spectra using the 5-nitroxy-derivative of stearic acid. Both approaches showed a decreased lipid fluidity in remnant-HDL₂, as compared to triglyceride-rich HDL₂. The immunoreactivity of apoA-I toward several monoclonal antibodies was assayed as a reflection of changes of apoA-I conformation. In remnant-HDL₂, as compared to triglyceride-rich HDL₂, a lower reactivity was noted with the 2G11 antibody, which interacts in the NH₂ terminal part of apoA-I. Finally, remnant-HDL₂ was clearly different from HDL₃ with respect to all of the parameters studied, demonstrating that hepatic lipase does not promote the direct conversion of HDL₂ to HDL₃. Thus, hepatic lipase produces remnant-HDL₂ particles, which display modifications of apoA-I conformation and of fluidity of the lipid environment. This newly described HDL₂ subfraction may play a major role in the reverse cholesterol transport.