The intima from aortas of normal New Zealand White rabbits was studied 2 hours after infusion of 320 mg human low density lipoprotein (LDL), resulting in a plasma concentration of at least five times and maximally 20 times the values found in normal rabbit serum. The following techniques were used: 1) ultrarapid freezing without chemical fixation, followed by freeze-etching; 2) immunofluorescence microscopy; and 3) postembedding immunogold-labeling electron microscopy. In the latter two methods MB47, a murine monoclonal antibody against human apolipoprotein B, was used as the primary antibody. Control rabbits were infused with the same volume of buffer only. Rotary-shadowed replicas of samples from the LDL-injected rabbits showed the deposition of lipidlike particles in the subendothelial-intimal space that were the size of the injected LDL (23 nm). In focal areas of the intima, groups of 23-nm-sized lipidlike particles and larger lipidlike structures were found enmeshed in the extracellular matrix. Control replicas were essentially free of lipid deposition. Immunofluorescence microscopy of frozen aortic cross sections showed an overall increase in apolipoprotein B in the intima of the LDL-injected rabbits. The presence of apolipoprotein B in the intima was also confirmed by immunoelectron microscopy. These in vivo results show that clustering of LDL-sized particles occurs in the intima within 2 hours of excessive LDL uptake. It also demonstrates the interaction of these LDL-sized particles with the filaments of the extracellular matrix. The clustering of the LDL-sized particles supports the possibility that LDL self-aggregation may occur in vivo and that components of the extracellular matrix are involved in this process.