The ability of migrating neurons and growth cones to navigate through their environment is crucial for the correct development of the brain. Cells and growth cones may be guided by electrical, chemical or topographical cues in their environment. Pax6 is a transcription factor vital for brain development. Pax6-/- mutant mice die perinatally with defects in neuronal proliferation and differentiation, cortical cell migration and axon guidance, yet it is not clear which guidance cues Pax6-/- mutant neurons fail to interpret. Dissociated cultured cells were used to study the cell-autonomous effects of Pax6 mutation on guidance of growth cones and migrating neural precursors by environmental cues. Neurites from mouse embryonic cortical neurons aligned perpendicular to 1 μm-wide, 130 nm-deep substratum grooves. Pax6-/- mutation abolished contact-mediated neurite guidance by these grooves. Laminin induced a switch from perpendicular to parallel alignment to grooves, via a β1 integrin-independent mechanism. Blocking cAMP signalling abolished perpendicular alignment to polylysine-coated grooves, but enhanced parallel alignment to laminin-coated grooves. Pax6 null mutation or overexpression also caused specific defects in contact-guided migration by cortical cells. An electric field applied to E16.5 cortical neurons increased the frequency of extension of neurites aligned perpendicular to the field axis. Pax6-/- mutant cells responded to an electric field with reduced anodal extension, but no significant increase in perpendicular neurite extension. Electrical cues were prioritised over topographical cues when presented in combination. Taken together, data suggest that Pax6 mutant cortical cells do not completely lack the ability to detect extracellular guidance cues, but they respond differently to wild-type cells. In combination with other defects identified in the cortex, this may contribute to the cell migration and axon guidance phenotypes in the brain of the Pax6-/- embryo. This study also identified novel Pax6 expression in the trigeminal ganglion, where it may regulate axon guidance and neurogenesis.