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To determine if magnetic resonance lymphography performed with subcutaneously administered AMI-227, a nanoparticulate iron oxide contrast agent, can distinguish reactive from tumor-bearing lymph nodes.Mature male Copenhagen rats were inoculated with cell suspensions of R3327-MAT-LyLu rat prostate carcinoma (n = 21) or Freund's complete adjuvant (n = 15) in the left footpad to generate ipsilateral popliteal lymph node metastases or lymphadenitis. At 12 to 14 days after inoculation, T1- and T2-weighted magnetic resonance images of bilateral popliteal areas were obtained before and 24 hours after subcutaneous administration of AMI-227. Contrast-to-noise ratios were calculated in precontrast and postcontrast images. Bilateral popliteal nodes were excised for pathologic assessment.AMI-227 resulted in decreased contrast-to-noise ratios in reactive (Tl − W = −7.01 ± 1.13, T2 − W = −31.64 ± 5.35) and normal (T1 − W = −13.56 ± 1.97, T2 − W = −21.62 ± 2.51) nodes. Contrast-to-noise ratios were unchanged (T1 − W = −0.22 ± 1.71, T2 − W = −2.20 ± 4.19) in tumor-containing nodes. These differences in contrast-to-noise ratio changes between tumor-bearing versus nontumor-bearing nodes were statistically significant (P < 0.05). Histologic analysis showed similar distribution of AMI-227 within normal and reactive nodes, but not in tumor-bearing nodes.Differences in AMI-227-uptake between tumor- and nontumor-bearing nodes detected with magnetic resonance imaging are helpful for distinguishing the two entities.