Immunogold labelling and electron microscopy were used to investigate β-1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye in situ. Molecular cytology allowed us to explore the hypothesis that this enzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore β-1,3-glucanase could play a role in pathogenicity. An extracellular endo-β-1,3-glucanase was purified from axenic culture and an antibody was raised. Enzyme activity staining and immunoblotting showed that the antibody was monospecific for β-1,3-glucanase present in fungal protein populations. Mycelia printings of plate-grown cultures displayed spot-like and streaky immuno-signals suggesting a secretion of β-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immuno gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one β-1,3-glucanase was immunoreactive. In inoculated plants, immunogold labelling was found in all infection stages and limited to the host-pathogen interface. Gold labelling was detected over fungal protoplasts in vacuoles and in tubular-vesicular complexes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of the fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of β-1,3-glucanase immuno-detected in infected ovaries. Antigenic sites were located in external, subcuticular, penetrating and intercellular hyphae, indicating that the enzyme was secreted throughout the colonization process in planta. Intense labelling regularly associated with fungal cell walls extended into adjacent host walls, indicating a migration of the fungal β-1,3-glucanase into the host apoplast. The gold labelling over host periplasmic spaces showed that the enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colonized inter- and intracellularly. Hyphae penetrated into the pectic middle lamella of sieve plates and intense immuno-labelling for β-1,3-glucanase in this area supports a phloem unblocking hypothesis.