During development, undifferentiated post-mitotic cells are integrated into increasingly complex tissues via cell-cell and cell-matrix interactions. The cell and extracellular matrix (ECM) interaction regulates many fundamental processes of development. Two recently discovered ECM molecules, netrin-4 and laminin γ3 chain, are expressed in the murine CNS. However, their expression and roles in the CNS have not been systemically studied. The working hypothesis is netrin-4 and laminin γ3 chain are important in murine CNS development, specifically in neuronal proliferation, adhesion, axon guidance and fasciculation. Using an anatomical and genetic approach, their spatial and temporal expression pattern and function in the CNS were examined. Netrin-4 is localized in both basement membrane (BM) and interstitial ECM; its expression in the interstitial ECM is developmentally regulated. The laminin γ3 chain is localized in various BMs with more limited distribution than the γ1 chain. In the netrin-4 null (Ntn4-/-) mice, BMs are not disrupted; no defects in pathfinding and only minor defects in RGC axon fasciculation are seen in the retina. The Ntn4-/- cerebella are considerably altered: granule cell (GC) number, the length of secondary fissures and the area of the internal granule cell layer (IGL) are significantly increased; there is additional lobulation. The proliferation of GC precursors (GCPs) is increased in Ntn4-/- mice as indicated by increased BrdU (+) cell density in the external germinal layer (EGL) at P6. In GC/GCP culture, application of exogenous netrin-4 decreases GCP proliferation in a concentration dependent manner. Together these data suggest that netrin-4 directly inhibits GCP proliferation. In the laminin γ3 chain null (Lamc3-/-) mice, BMs are not disrupted; minor morphological defects are observed in somatic tissues; no obvious defects are found in the retina. However, in the cerebellum, ectopic GCs and abnormal foliation pattern are observed, which are distinct from those described in the Ntn4-/- cerebellum. In retinal vascular BMs, netrin-4 and laminin γ3 chain are differentially distributed among arteries, veins and microvessels. In both Ntn4-/- and Lamc3-/- mice, microvascular branching may be increased. In conclusion, the expression of netrin-4 and laminin γ3 chain in the CNS is developmentally regulated; both molecules are important in cerebellar development.