Abstract In roots and mycorrhizae, the activity of phosphatases is an important parameter to characterise the efficiency of plants to access non-soluble phosphate pools in soils. We have quantified surface bound phosphatase (SBP) activity in non-mycorrhizal short roots and ectomycorrhizae of Nothofagus obliqua with image processed confocal laser scanning microscopy (LSM) using the fluorogenic substrate ELF-97 (enzyme-labelled fluorescence). Through interactive segmentation of root cells, mantle, and SBP-active centres, this method revealed a precise anatomical–physiological description of the SBP activity in cross-sections of short roots of N. obliqua, and of the mycorrhizal associations of N. obliqua with Pisolithus tinctorius and with Cenococcum geophilum in a controlled pH range (3–7). Our method revealed that the strategy of the examined species to vary the SBP activity was based primarily on the variation of the number and of the extension of the SBP-active centres. Fluctuations of the activities inside individual SBP-active centres were small. It was observed that non-mycorrhizal short roots of N. obliqua focus the distribution of SBP-active centres on the rhizodermis cells. In these cells, the SBP-active centres are distributed heterogeneously, and not preferentially in contact with the soil interface. The distribution of the SBP-active centres between the root cells and the mantle depended on the symbiont and on the pH of the buffer. The mantle hyphae of the N. obliqua– P. tinctorius associations promote direct contact between SBP-active centres and soil particles. In contrast, the mantle hyphae of N. obliqua– C. geophilum associations limit the expression of SBP-active centres to the interface between the mantle and the rhizodermis cells of N. obliqua. At this location, SBP-active centres are not in direct contact with any adjacent soil particles. Our observations of a pH-dependent activity, and of a mycorrhizal association-dependent activity of the SBP-active centres, together with the observed heterogeneity of the location of these centres relative to adjacent soil particles, challenge the general hypothesis that increased contact between mycorrhizae and soil results in higher efficiency of nutrient uptake.