MRI is being used increasingly for the noninvasive longitudinal monitoring of cellular processes in various pathophysiological conditions. Macrophages are the main stromal cells in neoplasms and have been suggested to be the major cell type ingesting superparamagnetic iron oxide (SPIO) nanoparticles. However, no MRI study has described longitudinally the presence of tumor-associated macrophages (TAMs) during tumorigenesis with histological confirmation. To address this, we injected SPIO nanoparticles into the circulation of tumor-bearing mice and used MRI and post-mortem histology to monitor TAMs at different time points. The MRI results demonstrated that TAMs, as hypointense signals, appeared continually with the expansion of the tumor. The histological findings also revealed that SPIO-labeled TAMs tended to deposit closer to the vessel lumen with time prior to rapid tumor growth. The present study demonstrates the potential of using MRI to assess longitudinally TAM accumulation during tumorigenesis, and provides the first in vivo insight into the topographical arrangement of TAMs in relation to the progression of tumors. In vivo monitoring of the presence of TAMs could be useful for the development of tumor treatments that target TAM functions. Copyright © 2011 John Wiley & Sons, Ltd.