Polysialic acid (polySia) is a posttranslational modification of the neural cell adhesion molecule NCAM, which in the vertebrate brain is dynamically regulated during development and crucially involved in developmental and adult neurogenesis. In the fish retina, new neurons are persistently generated, but the possible contribution of polySia has not yet been addressed. Here we used immunohistochemistry with NCAM‐ and polySia‐specific antibodies to study spatiotemporal expression patterns of NCAM and polySia in the developing and mature zebrafish retina. As early as 2.3 days postfertilization (dpf), NCAM but not polySia was detected on cell somata and fibers of the developing retina. At 4.3 dpf polySia immunoreactivity first appeared in the ventral retina and was localized to the nascent outer nuclear layer (ONL). In mature zebrafish, polySia immunoreactivity in the ONL extended to the entire retina. Colocalization with rhodopsin‐EGFP in transgenic zebrafish or the Müller glia‐specific protein cellular retinaldehyde‐binding protein (CRALBP) revealed that polySia immunoreactivity was confined to the compartment of radial Müller glia processes crossing the ONL and to a small band of processes positioned proximal to the horizontal cell layer of the mature retina. As shown by 5‐bromo‐2‐deoxyuridine (BrdU) labeling, both newly generated rod precursors within the mature ONL and precursors of the marginal zone were polySia‐negative. Thus, polySia‐negative rod precursors of the mature zebrafish retina face a polySia‐NCAM‐positive microenvironment presented by radial Müller glia. In view of the prominent role of polySia in other neurogenic systems, this pattern indicates that polySia provides environmental cues that are relevant for the generation of new rods. J. Comp. Neurol. 518:636–646, 2010. © 2009 Wiley‐Liss, Inc.