Two kinds of arginine deiminase (AD, EC 3.5.3.6) were purified from cell extracts of Mycoplasma arginini (a-AD) and Mycoplasma hominis (h-AD), and their enzymic properties and anti-tumor activities were compared. The a-AD enzyme strongly inhibited the growth of mouse hepatoma cell line MH134 in vitro, and its concentration required for 50% growth inhibition (IC50) was estimated to be about 10 ng/ml. The IC50 value of h-AD against the same cell line was estimated to be about 100 ng/ml, due to its low enzyme activity under the physiological pH condition, i.e., pH 7.4. These results show that the reaction pH profile of the a-AD was superior to that of the h-AD as an anti-tumor enzyme. Moreover, the effects of L-arginine metabolism-related substances on the anti-tumor activity of the a-AD were examined to study the growth-inhibitory mechanism of this enzyme. The addition of 2 or 4 mM L-arginine restored, in a dose-dependent manner, the growth of mouse MH134 hepatoma and Meth A fibrosarcoma cell lines that had been inhibited by 20 ng/ml of the a-AD. The addition of 2 or 4 mM L-ornithine, which is biosynthesized from L-arginine in the urea cycle and is the starting material in the polyamine-biosynthesis pathway, also partially restored it in a dose-dependent manner. These results indicate that the tumor cell growth inhibition caused by a-AD originates from the depletion of the essential nutrient L-arginine, and that the resulting block of the polyamine-biosynthesis pathway is involved in part in the inhibitory mechanism.