The ephrin-As, and their EphA receptor tyrosine kinases, guide retinal axons by contact-mediated repulsion to their correct target in the midbrain. We have developed a co-culture assay to observe the dynamic cytoskeletal rearrangements comprising retinal growth cone collapse stimulated by contact with an ephrin-A-expressing fibroblast. We show that EphA-ephrin-A interaction at membrane contact sites triggers rapid loss of growth cone lamellipodia followed by axon retraction and cell-cell separation. Using this assay, in combination with soluble ephrin-A5-induced growth cone collapse, we show that inhibiting the Rho effector, ROCK, prevents only ephrin-A-induced retinal axon retraction, but not loss of growth cone lamellipodia. This suggests that actin/myosin driven cell contraction alone does not mediate ephrin-A-induced repulsive responses. We provide evidence that Abl family kinases are a major effector of ephrin-A-induced retinal ganglion cell repulsion since the Abl inhibitor, STI571, prevents both loss of growth cone lamellipodia and axon retraction. These results comprise the first evidence that Abl family kinases play a role in EphA receptor-mediated axon guidance.