Laminar optogenetic functional magnetic resonance imaging (ofMRI), which combines high-resolution fMRI with layer-specific optogenetic stimulation, provides unprecedented insights into the functional organization of the neocortex and brain-wide networks. This technique allows for precise manipulation and observation of neural activity within specific cortical layers, offering valuable data on the distinct roles and interactions of these layers and remote brain regions. By elucidating the neural origins of laminar fMRI signals, we can enhance our understanding of brain-wide networks and the specific contributions of each cortical layer to overall brain function. This knowledge is crucial for developing targeted therapies for neurological disorders, potentially leading to more effective treatments for conditions, such as epilepsy, schizophrenia, and Alzheimer’s disease. In this chapter, the laminar ofMRI methods and examples are described. Transgenic mice expressing Cre-recombinase in specific neocortical layers and viral vectors to express channelrhodopsin-2 (ChR2) and enhanced yellow fluorescent protein (EYFP) (ChR2–EYFP) in these neurons were employed. Stereotaxic surgeries were performed to deliver the viral vectors and implant fiber-optic cannulas for optogenetic stimulation. Histological analyses validated the expression of ChR2. High-resolution fMRI was conducted to measure layer-specific cortical responses. It is foreseen that in the next decade, laminar ofMRI will contribute to unraveling several key aspects of cortical processing and brain-wide networks, transforming both basic neuroscience and clinical neurology research.