Since the commencement of functional magnetic resonance imaging (fMRI), great effort has been put into increasing its spatial resolution and signal specificity from vessel-weighted to more tissue-specific signals. The working assumption is that the "tissue" signals closely mirror changes at the neuronal level. While great progress has been made, the basic and most fundamental questions remain unanswered: where in the gray matter do these "tissue fMRI" changes occur? Recently, the temporal correspondence of hemodynamic-based fMRI signals and neurophysiological activity was explored. The data suggest, although not conclusively, that the local field potential (LFP) response gives a better estimate of changes that accompany increased neuronal activity. LFP are thought to be generated by synaptic activity reflecting input signals into layer IV within a cortical region. If so, the spatial distribution of the fMRI signal should be specific to the corresponding cortical lamina. Here, in a combined imaging and histological study, the spatial characteristics of fMRI signals across the lamina were explored. In a high-resolution fMRI study (0.15 × 0.15 × 2 mm3), the spatial specificity of fMRI signals was correlated with the underlying cortical laminar cytoarchitectonic obtained within the same animal and tissue region. We demonstrate that when surface vessels are excluded high-resolution fMRI signals peak at cortical layer IV.