Various macromolecular substances, such as toxins and antibodies, may interfere with neuromuscular transmission. The neuromuscular junction is also known to be a site for the uptake of macromolecular substances into the axon for subsequent transport to the central nervous system. The synaptic cleft of the neuromuscular junction is separated from the rest of the extracellular compartment of muscle by the basal lamina, the permeability properties of which are not known. The penetration of macromolecular substances of varying size into the synaptic cleft of the neuromuscular junction has been studied in rats. Four different tracers: Imferon (an iron-dextran measuring 11 X 7 X 7 nm), ferritin (a spherical iron-protein 12 nm in diameter), Imposil (an iron-dextran measuring 21 X 12 X 12 nm) and colloidal gold-protein (20-25 nm in diameter) were injected into the palmaris longus muscle. Fifteen and 120 min after injection, the distribution of these particulate tracers was studied by electron microscopy. Imferon and ferritin penetrated rapidly through the basal lamina along the muscle fibres and also into the synaptic cleft of the neuromuscular junction. The larger Imposil and colloidal gold particles were restricted from penetrating the basal lamina even after 2 h, and these particles were traced only occasionally within the synaptic cleft. The results indicate that the basal lamina of muscle acts as a diffusion barrier to large macromolecules, preventing them from entering the synaptic cleft.