<p>The optic tectum of the embryonic chick brain consists of 15 principal laminae arranged in a precise order and each with a unique identity. The transmembrane proteins ephrin-B1 and ephrin-B2 are both expressed during tectal neurogenesis. To investigate their role in establishing tectal lamination, we transfected RCAS-based, replication-competent retroviral expression vectors <i>in ovo</i> to ectopically express ephrin-Bs in the developing tectum, and analyzed the subsequent effect of ectopic domains of ephrin-B2 on tectal lamination and on the trajectories and laminar patterning of RGC axons. We found that ectopic expression of ephrin-B2 in developing tectum disrupted the uniform laminar organization in the developing tectum: e.g. tectal layers are distended coincident with domains of ephrin-B2 overexpression creating a ‘roller-coaster’-like effect. The laminar disturbance was not a result of increased or sustained cell proliferation or errors in tectal cell aggregation. Further, while tectal layer-specificity was maintained in transfection domains, afferent primary retinal ganglion cell axons circumvent ectopic domains of ephrin-B2 but appear unperturbed by ephrin-B1 domains, but were able to adjust to the undulations of tectal laminae, and terminate in the appropriate retinorecipient lamina. These findings demonstrate that ephrin-B2 is a repellent for RGC axons and has a unique role in the development of tectal lamination.</p>