Poly(amidoamine) (PAA) networks that are obtained by the use of cystamine as a cross-linking agent in the reaction with 2,2'-dithiodipyridine turn into linear PAAs with dithiopyridyl side groups that easily undergo an exchange reaction with thiocholesterol. The resultant products represent the first examples of amphiphilic PAA-cholesterol conjugates in which lipophilic cholesterol moieties are linked to the hydrophilic PAA chain by S-S bonds that are stable in blood but cleavable inside cells. In aqueous media, these conjugates self-assemble into nanoaggregates whose inner cores consist of lipophilic cholesterol domains. A series of PAA-cholesterol conjugates that are derived from two different bis-acrylamides, namely 2,2-bis(acrylamido) acetic acid and 1,4-bis(acryloyl)piperazine, and that have different cholesterol contents were obtained. All products were characterized by H-1 and C-13 NMR spectroscopy, and the average molecular weights of the soluble polymers were determined by size exclusion chromatography. In all instances, the segregation of cholesterol residues from the aqueous medium was revealed by the comparison of their NMR spectra in CDCl3 and D2O, respectively. The TEM analysis of the PAA-cholesterol aggregates in aqueous buffers revealed homogeneous round nanospheres whose dimensions and dimension distributions were determined by DLS. Preliminary cytocompatibility tests demonstrated that all prepared PAA-cholesterol samples are cytocompatible and thus show potential for biotechnological applications.