Changes in manganese-enhanced MRI (MEMRI) contrast across the rodent somatosensory cortex were compared to the cortical laminae as identified by tissue histology and administration of an anatomical tracer to cortex and thalamus. Across the cortical thickness, MEMRI signal intensity was low in layer I, increased in layer II, decreased in layer III until mid-layer IV, and increased again, peaking in layer V, before decreasing through layer VI. The reeler mouse mutant was used to confirm that the cortical alternation in MEMRI contrast was related to laminar architecture. Unlike in wild-type mice, the reeler cortex showed no appreciable changes in MEMRI signal, consistent with absence of cortical laminae in histological slides. The tract tracing ability of MEMRI was used to further confirm assignments and demonstrate laminar specificity. Twelve to 16 h after stereotaxic injections of MnCl(2) to the ventroposterior thalamic nuclei, an overall increase in signal intensity was detected in primary somatosensory cortex compared to other brain regions. Maximum intensity projection images revealed a distinctly bright stripe located 600-700 microm below the pial surface, in layer IV. The data show that both systemic and tract tracing forms of MEMRI are useful for studying laminar architecture in the brain.