The expression levels of the extracellular matrix glycoprotein laminin-1 are high along axonal tracts in the developing cerebellum, suggesting that laminin-1 plays a major role in cerebellar granule cell pathfinding. The purpose of this study was to determine the domain(s) of laminin-1 that confer its ability to guide axons and to investigate signaling molecules activated downstream of laminin-1 in the growth cone. We used a modified spot assay to quantify neurite behavior at a substrate interface between laminin-1 or the laminin-1 fragments E1', E4 and E8 and poly-L-lysine (PLL). We found that cerebellar granule cell neurites demonstrated a strong preference to remain on laminin-1 and the E8 fragment and to avoid the E1' fragment. Neurites exhibited random behavior at an interface between E4 and PLL. In addition, because members of the Rho GTPase family play an integral role in reorganization of the actin cytoskeleton, we evaluated whether neurite guidance at the laminin-1/PLL interface was impaired upon inhibiting Rho. The addition of C3 exoenzyme increased the percentage of neurites crossing from laminin-1 and E8 to PLL and from PLL to E1'. Furthermore, inhibiting Rho did not alter neurite lengths on laminin-1, E1', E4 or E8, demonstrating a requirement for Rho activation in neurite guidance but not in neurite outgrowth. These results suggest that Rho activation is essential for growth cone turning at a laminin-1 boundary.