Patterns of coordinated spontaneous activity have been proposed to guide circuit refinement in many parts of the developing nervous system. It is unclear, however, how such patterns, which are thought to indiscriminately synchronize nearby cells, could provide the cues necessary to segregate functionally distinct circuits within overlapping cell populations. Here, we report that glutamatergic retinal waves possess a substructure in the bursting of neighboring retinal ganglion cells with opposite light responses (ON or OFF). Within a wave, cells fire repetitive nonoverlapping bursts in a fixed order: ON before OFF. This pattern is absent from cholinergic waves, which precede glutamate-dependent activity, providing a developmental sequence of distinct activity-encoded cues. Asynchronous bursting of ON and OFF retinal ganglion cells depends on inhibition between these parallel pathways. Similar asynchronous activity patterns could arise throughout the nervous system, as inhibition matures and might help to separate connections of functionally distinct subnetworks.