The neocortex is known to have a distinct laminar structure which has previously been probed in animals using high-resolution fMRI. Detection of layer-specific activation in humans has however to date proven elusive. In this study we demonstrate for the first time such layer-specific activation, specifically at a depth corresponding to layer IV of human primary visual cortex (V1). We used a gradient-echo (GE) sequence at 3T with an isotropic resolution of 0.75 mm, in which a stria at the depth of layer IV was visible in the averaged time series, and could be used as an anatomical landmark. Upon visual stimulation (7.5 Hz flickering checkerboard) the signal increase of 3% in layer IV was significantly higher than in the neighboring laminae. The width of this activation peak was 0.8-1 mm. Based on this result and known laminar organization of the intracortical vasculature we conclude that in the direction perpendicular to the cortical surface the intrinsic spatial resolution of the GE-BOLD fMRI signal is in the submillimetre range. Human laminar fMRI is a significant development which may improve our understanding of intracortical activation patterns and of the way in which different cortical regions interact.