Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. Here we describe a role for a Drosophila Eph receptor tyrosine kinase (EPH) in the control of photoreceptor axon and cortical axon topography in the developing visual system. Although uniform across the developing eye, EPH is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of EPH activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbed the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons resulted from the eye-specific expression of the dominant-negative EPH transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates.