Developing chick retinotectal projections extend rostrally in the superficial stratum opticum of the tectum until they reach their appropriate target zone. They then penetrate, arborize, and form synapses within distinct tectal retinorecipient layers. In this study, we show that the polysialylated neural cell adhesion molecule is expressed both on the membrane of these developing projections and in the stratum opticum and retinorecipient layers during the period of optic innervation. On this basis, the role of polysialic acid was analyzed with respect to both trajectory and arborization in the tectum, using confocal imaging of DiI-labeled retinotectal fibers in whole-mount tecta of embryos pretreated with a polysialic acid-specific degrading enzyme, endoneuraminidase N. The removal of polysialic acid caused several distinct abnormalities, including random dorsal/ventral meandering of fibers in the stratum opticum, a distorted branching and extension of arbors in the retinorecipient layers, and inappropriate synaptic vesicle accumulation in pretarget areas. These findings indicate that the unique ability of polysialic acid to regulate different types of cell interactions is an essential component of axon behavior during multiple steps of tectal target innervation. J. Comp. Neurol. 460:203–211, 2003. © 2003 Wiley-Liss, Inc.