Visualization of the protein secretory pathway in the high secreting trichoderma reesei (T. reesei) hyphae using confocal microscopy will lead to better understanding of the cellular mechanisms involved in protein secretion and contribute to the identification of bottlenecks in the secretion of foreign proteins from fungi. An introduction into visualization approaches involved the application of the fluorescent dye FM 4-64 for staining of membrane-based structures in T. reesei hyphae. Confocal microscopy studies were carried out with 24-hour old cultures of the T. reesei strain Rut-C30. Staining of the fungal hyphae and characteristics of FM 4-64 including spectral properties, time course of the labeling and double labeling with other specific organelle dyes will be discussed.