A number of kinetic properties of the negative and neutral arginine kinases isolated from the horseshoe crab, Limulus polyphemus, were studied. The two arginine kinase forms show very similar kinetic properties. Both require a divalent cation such as Mg 2+ or Mn 2+ for activity. Ca 2+ is about 40% as active as Mg 2+ while Zn 2+, Cu 2+, Fe 2+ and Fe 3+ are not activators. High concentrations of free Mg 2+ will inhibit both enzymes. Optimal enzyme activity is observed in the forward reaction when the concentration of free Mg + is 1 m m. Other nucleoside triphosphates can substitute for ATP as substrates in the forward reaction. The order of decreasing V is ATP > 2′dATP ⋙ ITP > GTP for both forms. Neither d-arginine nor l-argininic acid will act as substrates for either isoenzyme, but both compounds are competitive inhibitors with respect, to l-arginine. l-Canavanine is a substrate for both forms, but its optimal velocity is much less than that observed with l-arginine. l-Canavanine shows sigmoid kinetics with both forms. The results of initial velocity studies of the forward reaction indicate that the mechanism for both forms is of the random order rapid equilibrium type similar to that observed with rabbit muscle creatine kinase and Panulirus longipes arginine kinase. Isotope-exchange measurements made with the negative form were also consistent with this mechanism. Although no catalytic basis for a functional difference between the two forms was found, it is possible that in vitro differences in stability may reflect stabilities in vivo and thus provide a basis for regulating arginine kinase levels in various tissues thereby giving a selective advantage to species having both forms.