The apoplasmic permeability of ectomycorrhizal roots of intact Eucalyptus pilularis seedlings infected with Pisolithus tinctorius on aseptic agar plates was examined using the nonbinding fluorochrome 8-hydroxypyrene-1,3,6-trisulphonate and lanthanum ions in conjunction with anhydrous freeze substitution and dry sectioning. Most mycorrhizas formed in the air above the agar surface, and in these the sheath rapidly became nonwettable and impermeable to the fluorochrome but was nevertheless permeable to lanthanum ions. In a few mycorrhizas which developed in contact with the agar the sheath remained permeable to both tracers when fully developed. This increased hydrophobicity of the sheath in mycorrhizas in the air above the agar surface might be explained by deposition of hydrophobins, but nevertheless it still allows an apoplasmic pathway for radial movement of ions. Regardless of their sheath permeation both apoplasmic tracers were always found throughout the Hartig net and were arrested at the Casparian bands and suberin lamellae of the exodermis. It is concluded that the fluorochrome must have moved longitudinally along the Hartig net which is a region of higher permeability than the sheath. Casparian bands in the exodermis of ectomycorrhizal roots have similar properties to those in nonmycorrhizal roots in excluding solutes and their exclusion of lanthanum ions indicates that they are not permeable to ions. The data do not support the concept of a totally sealed apoplasmic exchange compartment, but the differential permeability suggests that the sheath might allow radial transfer of ions but block loss of sugars and organic molecules of similar size.