Substrate-bound guidance cues play an important role during the development of thalamocortical projections. We used time-lapse video microscopy to study the growth behaviour of thalamic axons on different substrates. On embryonic cortical membranes and on a pure laminin substrate, thalamic fibres advanced relatively slowly (∼15 μm/h) and on average their growth cones retracted transiently every ∼5 h. In contrast, on membranes prepared from early postnatal cortex, thalamic fibres grew twice as fast and spontaneous growth cone collapse occurred ∼8 times less often. Experiments in which we used the sugar-binding lectin peanut agglutinin or heat inactivation to change the membrane properties indicated that these differences are due to growth-supporting molecules on postnatal cortical membranes. When offered a choice between embryonic and postnatal cortical membranes, thalamic axons preferred the postnatal membrane substrate. Time-lapse imaging revealed that borders between these two substrates effectively guided thalamic fibres, and in most cases axons changed their direction without collapse of the growth cone. Our results suggest that thalamic axons can be guided by the spatial distribution of growth-promoting molecules in the developing cortex.