The six cortical layers have distinct anatomical and physiological properties, like different energy use and different feedforward and feedback connectivity. It is not known if and how layer-specific neural processes are reflected in the fMRI signal. To address this question we used high-resolution fMRI to measure BOLD, CBV, and CBF responses to stimuli that elicit positive and negative BOLD signals in macaque primary visual cortex. We found that regions with positive BOLD responses had parallel increases in CBV and CBF, whereas areas with negative BOLD responses showed a decrease in CBF but an increase in CBV. For positive BOLD responses, CBF and CBV increased in the center of the cortex, but for negative BOLD responses, CBF decreased superficially while CBV increased in the center. Our findings suggest different mechanisms for neurovascular coupling for BOLD increases and decreases, as well as laminar differences in neurovascular coupling. ► High-resolution fMRI allows visualization of laminar differences in BOLD, CBV, and CBF ► CBF is decreased for negative BOLD while CBV is increased ► Positive and negative BOLD signals have different neurovascular coupling mechanisms ► Neurovascular coupling differs depending on cortical depth Goense et al. use high-resolution fMRI to show that positive and negative BOLD responses have different neurovascular coupling mechanisms and that neurovascular coupling is cortical layer dependent. This potentially allows disentangling of feedforward, feedback, excitatory, or inhibitory processes using fMRI.