The retinocollicular projection is a preferred axon guidance pathway for investigating molecular mechanisms of synaptic targeting in the mammalian CNS. Here we identify a previously unrecognized role of the L1 cell adhesion molecule in topographic mapping of retinal ganglion cell (RGC) axons to their targets in the mouse superior colliculus (SC). L1 was transiently expressed on RGC axons during axon growth and targeting. DiI labeling of retinal axons revealed that temporal axons of L1-minus mice bypassed correct target locations in the anterior SC, forming termination zones at incorrect posterior sites, which were often skewed along the mediolateral axis. During development of the retinotopic map L1-minus temporal axons extended across the anteroposterior axis of the SC like wild-type axons but failed to arborize at normal anterior target sites. L1-minus RGC axons exhibited normal crossing at the optic chiasm and fasciculation of the optic nerve. Results suggest that retinal axons require the function of L1 in addition to repellent EphA guidance receptors to achieve proper topographic mapping.