Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Müller glia (MG) cells. We demonstrate that the Crb1-/- phenotype, as shown in Crb1-/- mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1-/- primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. We conclude that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells.