A pronounced effect of concanavalin A (Con A) upon activity of ecto-5'-nucleotidase of intact C6 glioma cells in culture has been demonstrated. A near linear rate of decrease in 5'-nucleotidase activity was observed upon treatment with concentrations of Con A up to 0.25 muM. Nonspecific phosphatase activity and Ca2+-dependent ATPase activity were not inhibited by Con A treatment of the cells. Of the total 5'-nucleotidase activity of C6 cells (Vmax = 5.0 mumol of Pi liberated/mg of cell protein/hour), approximately 20% still remained after treatment with high concentrations of Con A. The inhibitory effect of Con A operated to reduce substantially Vmax for ecto-5'-nucleotidase. Inhibition was reversed by briefly incubating the Con A-treated cells with alpha-methyl-D-glucoside, or alpha-methyl-D-mannoside, the later being more effective. These findings suggest that a relatively specific, reversible, inhibition of ecto-5'-nucleotidase results from Con A binding to the surface of the intact cultured mammalian cells.