The formation of the olivocerebellar projection is supposed to be regulated by positional information shared between pre- and postsynaptic neurons. However, experimental evidence to support this hypothesis is missing. In the chick, caudal neurons in the inferior olive project to the anterior cerebellum and rostral ones to the posterior cerebellum. We here report in vitro experiments that strongly support the existence of anteroposterior polarity cues in the embryonic cerebellum. We developed an in vitro system that was easily accessible to experimental manipulations. Large hindbrain explants of E7.5-E8 chick embryos, containing the cerebellum and its attached brainstem, were plated and studied using axonal tracing methods. In these cultures, we have shown that the normal anteroposterior topography of the olivocerebellar projection was acquired, even when the cerebellar lamella was detached from the brainstem and placed again in its original position. We also found that, following various experimental rotations of the anteroposterior axis of the cerebellum, the rostromedian olivary neurons still project to the posterior vermis and the caudolateral neurons to the anterior vermis, that now have inverted locations. Thus, the rotation of the target region results in the rotation of the projection. In addition, we have shown that the formation of the projection map could be due to the inability of rostromedian inferior olivary axons to grow in the anterior cerebellum. All these experiments strongly indicate that olivocerebellar fibers recognize within their target region polarity cues that organize their anteroposterior topography, and we suggest that Purkinje cells might carry these cues.