Detailed 3D mouse brain images may promote better understanding of phenotypical differences between normal and transgenic/mutant mouse models. Previously, a number of magnetic resonance microscopy (MRM) studies have successfully established brain atlases, revealing genotypic traits of several commonly used mouse strains. In such studies, MR contrast agents, mainly gadolinium (Gd) based, were often used to reduce acquisition time and improve signal-to-noise ratio (SNR). In this paper, we intended to extend the utility of contrast agents for MRM applications. Using Gd-DTPA and MnCl(2), we exploited the potential use of MR contrast agents to manipulate image contrast by drawing upon the multiple relaxation mechanisms and tissue-dependent staining properties characteristic of each contrast agent. We quantified r(1) and r(2) of Gd-DTPA and MnCl(2) in both aqueous solution and brain tissue and demonstrated the presence of divergent relaxation mechanisms between solution and tissue for each contrast agent. Further analyses using nuclear magnetic resonance dispersion (NMRD) of Mn(2+) in ex vivo tissue strongly suggested macromolecule binding of Mn(2+), leading to increased T(1) relaxation. Moreover, inductively coupled plasma (ICP) mass spectroscopy revealed that MnCl(2) had higher tissue affinity than Gd-DTPA. As a result, multiple regions of the brain stained by the two agents exhibited different image contrasts. Our results show that differential MRM staining can be achieved using multiple MR contrast agents, revealing detailed cytoarchitecture, and may ultimately offer a window for investigating new techniques by which to understand biophysical MR relaxation mechanisms and perhaps to visualize tissue anomalies even at the molecular level.