Information about layer specific connections in the brain comes mainly from classical neuronal tracers that rely on histology. Manganese Enhanced MRI (MEMRI) has mapped connectivity along a number of brain pathways in several animal models. It is not clear at what level of specificity neuronal connectivity measured using MEMRI tracing can resolve. The goal of this work was to determine if neural tracing using MEMRI could distinguish layer inputs of major pathways of the cortex. To accomplish this, tracing was performed between hemispheres of the somatosensory (S1) cortex and between the thalamus and S1 cortex. T(1) mapping and T(1) weighted pulse sequences detected layer specific tracing after local injection of MnCl(2). Approximately 12 h following injections into S1 cortex, maximal T(1) reductions were observed at 0.6+/-0.07 and 1.1+/-0.12 mm from the brain surface in the contralateral S1. These distances correspond to the positions of layer 3 and 5 consistent with the known callosal inputs along this pathway. Four to six hours following injection of MnCl(2) into the thalamus there were maximal T(1) reductions between 0.7+/-0.08 and 0.8+/-0.08 mm from the surface of the brain, which corresponds to layer 4. This is consistent with terminations of the known thalamocortical projections. In order to observe the first synapse projection, it was critical to perform MRI at the right time after injections to detect layer specificity with MEMRI. At later time points, tracing through the cortical network led to more uniform contrast throughout the cortex due to its complex neuronal connections. These results are consistent with well established neuronal pathways within the somatosensory cortex and demonstrate that layer specific somatosensory connections can be detected in vivo using MEMRI.