Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.