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In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin\u2010B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin\u2010B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin\u2010B2, we utilized time\u2010lapse imaging to characterize the effects of ephrin\u2010B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin\u2010B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally\u2010projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally\u2010projecting ventronasal axons are less sensitive to ephrin\u2010B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general. \u00a9 2010 Wiley Periodicals, Inc. 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