The mechanisms underlying the precise targeting of tectal layers by ingrowing retinal axons are largely unknown. In zebrafish, individual axons choose one of four retinorecipient layers upon entering the tectum and remain restricted to this layer, despite continual remodeling and shifting of their terminal arbors. In dragnet mutants, by contrast, a large fraction of retinal axons aberrantly trespass between layers or form terminal arbors that span two layers. The dragnet gene, drg, encodes collagen IV(alpha5) (Col4a5), a basement membrane component lining the surface of the tectum. Heparan sulfate proteoglycans (HSPGs) are normally associated with the tectal basement membrane but are dispersed in the dragnet mutant tectum. Zebrafish boxer (extl3) mutants, which are deficient in HSPG synthesis, show laminar targeting defects similar to those in dragnet. Our results show that the collagen IV sheet anchors secreted factors at the surface of the tectum, which serve as guidance cues for retinal axons.