The location and activity of saprophytic and arbuscular mycorrhizal (NM) fungal phosphatases have been investigated with a fluorogenic substrate (ELF) that forms a fluorescent crystalline precipitate at the site of phosphatase activity. This ELF substrate method was compared with other detection methods such as fast blue RR salt staining and p-nitrophenyl phosphate. The ELF substrate was successfully used to determine the location of phosphatase activity in AM fungal hyphae, and to visualize the active AM fungal colonization of roots. The substrate was taken up by fungal hyphae, when acid and alkaline buffers were used, and internal phosphatase activity was visualized. The release of both acid and alkaline phosphatases by a saprophytic fungus to an agar medium was demonstrated. ELF combined with an alkaline buffer led to specific staining of active fungal structures such as hyphae, arbuscules and young vesicles in AM-colonized roots. The results indicate that the ELF substrate is a sensitive indicator for fungal phosphatase activity and that it can be used to visualize hyphal phosphatase activity in both roots and external hyphae.