The three-dimensional shapes and sizes of plasma lipoproteins and astrocyte-secreted lipoproteins (ASLPs) were characterized with the aid of in situ atomic force microscopy (AFM), which has the unique ability to study three-dimensional nanostructures under physiological conditions. Apolipoprotein E (apoE) and apolipoprotein J (apoJ) are the two most abundant apolipoproteins produced in the central nervous system (CNS). This study revealed that ASLPs containing apoE3, apoE4, or apoJ significantly differ from high density lipoprotein particles, thought to be their closest analogs in plasma, in aggregation properties, size, and shape. ASLPs were found to be significantly flatter and smaller than their plasma counterparts. Plasma lipoproteins were able to form ordered arrays on a mica surface at high concentration, but ASLPs did not. Rather, they formed amorphous aggregates at similar concentrations. Comprehensive quantitative characterization of particle size and shape was facilitated by two advances in AFM image analysis: (1) automated analysis through image-recognition algorithms, and (2) correction for the finite size of the AFM probe based on geometric modeling. This study and the developed AFM methodologies open the way to further in situ AFM studies of the lipoproteins in general and more specifically of CNS lipoproteins.