The colonization of plant roots by mycorrhizal fungi, generally increases P content in the host plant when in soils with low levels of P. The objective of this work was to evaluate the activity of acid phosphatases in two isolates of Pisolithus microcarpus, grown in different sources and concentrations of phosphate and to characterize the phosphatase isoenzymes produced by isolates. Both isolates were grown on Melin-Norkrans modified (MNM) medium enriched with organic (Po) or inorganic (Pi) sources of phosphorus, at five different concentrations, in order to study the activity of mycelial surface acid phosphatases during incubation for up to 96 h. Activity of acid phosphatases increased when the fungi were grown without or at low concentrations of Pi. Intraspecific differences were observed between the isolates with regard to acid phosphatase production. A greater decrease in phosphatase activity was observed when incubation time was increased than when Pi concentration was increased. At an incubation time of 96 h, activity of acid phosphatases in isolate 90A increased with increasing Po concentration, while for isolate RV82 remained constant over the different incubation periods and Po concentrations tested. When grown in media without Pi, an additional band appeared in the isoenzyme pattern of RV82, while isoenzyme production was not altered in isolate 90A when grown in media without Pi or with 2 mM Pi, showing differences in their isoenzyme patterns. Isolate 90A possesses a potential competitive advantage when utilized in mycorrhizal association with Eucalyptus because of its ability to utilize Po.