To clarify the roles of extracellular matrix molecules (ECMs) and their receptors, integrins, in axonal pathfinding, their spatiotemporal localization was studied immunohistochemically in the developing mouse spinal cord. Immunoreactivities for collagen type IV (Col IV), laminin, integrins (alpha2, alpha3 and beta1 subunits), and transforming growth factor (TGF) betas (TGF beta2 and TGF beta3), putative regulators of gene expression of these molecules, were localized on the midline of the ventral spinal cord, or the 'floor plate', in normal ICR mice. These immunoreactions appeared at embryonic day 9 (E9), when commissural axons began to cross the floor plate, and peaked at E13-E14 when more axons crossed, turned rostrally, and projected longitudinally. At this stage, TAG-1-immunopositive commissural axons passed through the floor plate region. Longitudinal axons, on the other hand, projected ipsilaterally parallel to the midline. Immunoreactions for ECMs, integrins and TGF betas were weakly positive at the midline until postnatal day 0 (P0), after which they decreased markedly. In Danforth's short tail (Sd) heterozygous mutants with discontinuous floor plates, immunoreactions for ECMs, integrins, and TGF betas were not detected at P0. Few crossing axons were seen at this stage, and longitudinally ascending axons crossed via aberrant paths in these regions. These results suggest that the restricted localization of ECMs, integrins, and TGF betas at the floor plate is involved in the pathfinding of commissural and longitudinal axons.