The ability to encode the direction of image motion is fundamental to our sense of vision. Direction selectivity along the four cardinal directions is thought to originate in direction-selective ganglion cells (DSGCs) because of directionally tuned GABAergic suppression by starburst cells. Here, by utilizing two-photon glutamate imaging to measure synaptic release, we reveal that direction selectivity along all four directions arises earlier than expected at bipolar cell outputs. Individual bipolar cells contained four distinct populations of axon terminal boutons with different preferred directions. We further show that this bouton-specific tuning relies on cholinergic excitation from starburst cells and GABAergic inhibition from wide-field amacrine cells. DSGCs received both tuned directionally aligned inputs and untuned inputs from among heterogeneously tuned glutamatergic bouton populations. Thus, directional tuning in the excitatory visual pathway is incrementally refined at the bipolar cell axon terminals and their recipient DSGC dendrites by two different neurotransmitters co-released from starburst cells.