THE subcellular interactions of the neuronal microtubule-associated proteins tau, MAP2 and MAP5 were determined in cultured rat hippocampal neurons using differential detergent extraction and laser scanning microscopy. Axon development correlated with a transition from a MAP2-actin filament interaction to a MAP2-microtubule association and binding of tau to the distal axon. Tau and MAP2 binding specifically increased in the axon and the minor neurites, respectively. No compartment-specific association of MAP5 was observed. Tau binding preceded the accumulation of microtubules at the distal axon which represented a characteristic event during axogenesis. The data provide evidence for a role of MAP2 in regulating microfilament-microtubule interactions during neurite formation and of tau in organizing microtubules at the distal axon.