The use of manganese ions (Mn(2+)) as an MRI contrast agent was introduced over 20 years ago in studies of Mn(2+) toxicity in anesthetized rats (1). Manganese-enhanced MRI (MEMRI) evolved in the late nineties when Koretsky and associates pioneered the use of MEMRI for brain activity measurements (2) as well as neuronal tract tracing (3). Currently, MEMRI has three primary applications in biological systems: (1) contrast enhancement for anatomical detail, (2) activity-dependent assessment and (3) tracing of neuronal connections or tract tracing. MEMRI relies upon the following three main properties of Mn(2+): (1) it is a paramagnetic ion that shortens the spin lattice relaxation time constant (T(1)) of tissues, where it accumulates and hence functions as an excellent T(1) contrast agent; (2) it is a calcium (Ca(2+)) analog that can enter excitable cells, such as neurons and cardiac cells via voltage-gated Ca(2+) channels; and (3) once in the cells Mn(2+) can be transported along axons by microtubule-dependent axonal transport and can also cross synapses trans-synaptically to neighboring neurons. This chapter will emphasize the methodological approaches towards the use of MEMRI in biological systems.