A phosphatase activity of the trypanosomatid parasite Herpetomonas samuelpessoai was characterized using intact living cells. The effects of dimethyl sulfoxide (DMSO) on this activity were investigated. This phosphatase activity (2.53+/-0.01 nmol P(i)/mg protein x min) was linear with cell density and with time for at least 60 min. The optimum pH for the H. samuelpessoai phosphatase lies in the acid range. This phosphatase activity was inhibited by metal chelators and classical phosphatase inhibitors. A robust stimulation of the phosphatase activity was observed when the flagellates were grown in the presence of 4% DMSO, both when intact flagellates and when culture supernatant from those cells were assayed, as observed by biochemical and cytochemical analysis. We also demonstrate that DMSO induced the secretion and/or shedding of this phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process.