Layer-dependent fMRI allows measurements of information flow in cortical circuits, as afferent and efferent connections terminate in different cortical layers. However, it is unknown to what level human fMRI is specific and sensitive enough to reveal directional functional activity across layers. To answer this question, we developed acquisition and analysis methods for blood-oxygen-level-dependent (BOLD) and cerebral-blood-volume (CBV)-based laminar fMRI and used these to discriminate four different tasks in the human motor cortex (M1). In agreement with anatomical data from animal studies, we found evidence for somatosensory and premotor input in superficial layers of M1 and for cortico-spinal motor output in deep layers. Laminar resting-state fMRI showed directional functional connectivity of M1 with somatosensory and premotor areas. Our findings demonstrate that CBV-fMRI can be used to investigate cortical activity in humans with unprecedented detail, allowing investigations of information flow between brain regions and outperforming conventional BOLD results that are often buried under vascular biases. •A CBV-fMRI method is developed for sampling brain activity across cortical layers•We can separately measure input and output activity of primary motor cortex•CBV-fMRI can measure laminar activity with higher accuracy than BOLD-fMRI•Laminar resting-state fMRI can reveal directional connectivity between brain areas Huber et al. demonstrate an MRI method to measure brain activity changes at the spatial resolution of cortical layers in humans. This allows investigations of directional functional connectivity, paving the way for non-invasive studies investigating information flow between brain regions.