Feedforward connections are thought to be important in the generation of orientation-selective responses in visual cortex by establishing a bias in the sampling of information from regions of visual space that lie along a neuron's axis of preferred orientation. It remains unclear, however, which structural elements-dendrites or axons-are ultimately responsible for conveying this sampling bias. To explore this question, we have examined the spatial arrangement of feedforward axonal connections that link non-oriented neurons in layer 4 and orientation-selective neurons in layer 2/3 of visual cortex in the tree shrew. Target sites of labeled boutons in layer 2/3 resulting from focal injections of biocytin in layer 4 show an orientation-specific axial bias that is sufficient to confer orientation tuning to layer 2/3 neurons. We conclude that the anisotropic arrangement of axon terminals is the principal source of the orientation bias contributed by feedforward connections.