Co-culture of the pathogenic fungus Fusarium oxysporum f. sp. pini Schlecht. emend Snyd. & Hans, with the ectomycorrhizal fungus Paxillus involutus Fr. on potato dextrose agar caused no visible morphological changes in the fungi. Co-culture on modified Melin Norkrans medium in petri plates yielded, however, protoplast formation at or near tips of P. involutus hyphae. No changes were visible in F. oxysporum as a result of co-culture and the two fungi grew in close association with each other without inhibition. Staining with Calcofluor White M2R New and fluorescein diacetate displayed that, at this stage in development, a wall surrounding the protoplast was absent but a plasma membrane sheathed by an extracellular mucilage was present. Protoplasts lacking definite form adhered closely to adjoining hyphae while those with the characteristic spherical form were easily dislodged from hyphae. Average size of spherical protoplasts was 17–20 μm. Breaks were present in the hyphal wall in regions adjacent to protoplast formation and these may have been pathways for cytoplasmic leakage. Transmission electron microscopy revealed these breaks more clearly, and provided additional evidence of a plasma membrane around protoplast cytoplasm. Protoplasts contained few organelles except vesicles, larger electron dense bodies and occasional nuclei. Hyphae forming protoplasts often lacked cytoplasmic integrity although those not forming protoplasts appeared healthy.