The purpose of this study was to synthesize biocompatible poly(2-hydroxyethyl aspartamide)–C 16 -iron oxide (PHEA-C 16 -iron oxide) nanoparticles and to evaluate their efficacy as a contrast agent for magnetic resonance imaging of lymph nodes. The PHEA-C 16 -iron oxide nanoparticles were synthesized by coprecipitation method. The core size of the PHEA-C 16 -iron oxide nanoparticles was about 5 to 7 nm, and the overall size of the nanoparticles was around 20, 60, and 150 nm in aqueous solution. The size of the nanoparticles was controlled by the amount of C 16 . The 3.0-T MRI signal intensity of a rabbit lymph node was effectively reduced after intravenous administration of PHEA-C 16 -iron oxide with the size of 20 nm. The in vitro and in vivo toxicity tests revealed the high biocompatibility of PHEA-C 16 -iron oxide nanoparticles. Therefore, PHEA-C 16 -iron oxide nanoparticles with 20-nm size can be potentially useful as T2-weighted MR imaging contrast agents for the detection of lymph nodes.