During atherogenesis, lipid droplets appear in the extracellular space of the arterial intima. We previously observed generation of lipid droplets on the surface of exocytosed mast cell granules when granule neutral proteases degraded the granule-bound LDL particles and the particles became unstable and fused [Kovanen, P.T., & Kokkonen, J.O. (1991) J. Biol. Chem. 266, 4430-4436]. We have now extended our studies to the fluid phase and examined the effects of several proteases (trypsin, alpha-chymotrypsin, Pronase, plasmin, kallikrein, and thrombin) all known for their ability to cleave the apolipoprotein B-100 component (apoB-100) of LDL. The fused LDL particles were separated from unfused particles by gel filtration or by density gradient ultracentrifugation. Proteolytic degradation of LDL with trypsin, alpha-chymotrypsin, or Pronase led to fragmentation of apoB-100 and release of the fragments from the LDL particles and triggered particle fusion. In contrast, proteolytic degradation of LDL with plasmin, kallikrein, or thrombin, which also led to fragmentation of apoB-100 but not to release of fragments, did not trigger particle fusion. With advancing degradation of apoB-100, particles having progressively lower densities and larger sizes were generated. Thus, after incubation for 24 h with alpha-chymotrypsin (apoB-100:alpha-chymotrypsin mass ratio 10:1) 40% of the apoB-100 was degraded and about 30% of the LDL particles had fused and reached diameters of up to 70 nm and densities ranging from 1.020 to < 1.005 g/mL. When the proteolyzed LDL particles, both unfused and fused, were incubated with macrophages, only those particles that had undergone fusion were ingested and converted into intracellular cholesteryl ester droplets. Thus proteolysis of LDL with release of apoB-100 fragments renders the particles sufficiently unstable to fuse and thus to become liable to ingestion by macrophages. Since the fused LDL particles resemble the extracellular lipid droplets in the atherosclerotic arterial intima and generate foam cells in vitro, these findings support the idea that proteolytic fusion of LDL is an atherogenic process.