Extraradical hyphae of Glomus intraradices or G. claroideum were extracted from root free sand of two-compartment pot cultures and used to determine fungal phosphatase activity [p-nitrophenyl phosphate (p-NPP) hydrolysis]. Enzyme activity was assayed with respect to pH, temperature and different fractions of the hyphae (external soluble, wall-bound and internal phosphatases). The results showed an overall maximum enzyme activity at pH 5·2–5·6 for both fungi, with a possible secondary maximum at pH [ges ]8·8 for G. claroideum. Of the two fungi tested, G. intraradices had the highest external phosphatase activity in two experiments and the same activity in one experiment, reaching 184 μmol p-NPP hydrolysed mg−1D.W. h−1. Phosphatase activity at pH 5·2 decreased sharply with temperature, with 4·5 and 10·5% of the enzyme activity remaining at 5 °C relative to that at 37° for G. intraradices and G. claroideum, respectively. Separation of the phosphatase activity into external soluble, wall-bound and internal fractions revealed that up to 70% of the measured activity was associated with the hyphal wall, and the rest with internal structures. Exuded phosphatases were not found in measurable amounts. The implications of these results on possible hyphal utilization of organic P in soil are discussed.