Knowledge of the kinetics of the rod cyclic GMP phosphodiesterase is essential for understanding the kinetics and gain of the light response. Therefore, the interactions between Mg2+, cyclic GMP, and purified, trypsin-activated bovine rod cyclic GMP phosphodiesterase (EC 18.104.22.168) were examined. The effects of Mg2+ and of cyclic GMP on the rod phosphodiesterase activity were mutually concentration-dependent. Formation of a free Mg-cyclic GMP complex is unlikely due to its high dissociation constant (Kd = 19 mM). Plots of 1/velocity versus 1/[cyclic GMP] as a function of [Mg2+] and 1/velocity versus 1/[Mg2+] as a function of [cyclic GMP] intersected to the left of the 1/velocity axis. This is consistent with the formation of a ternary complex between the phosphodiesterase, Mg2+, and cyclic GMP. A competitive inhibitor of the phosphodiesterase relative to cyclic GMP, 3-isobutyl-1-methylxanthine, non-competitively inhibited the enzyme relative to Mg2+, Pb2+, a competitive inhibitor of the phosphodiesterase relative to Mg2+ [D. Srivastava, R.L. Hurwitz and D. A. Fox (1995) Toxicol. Appl. Pharmacol, in the press] non-competitively inhibited the enzyme relative to cyclic GMP. Collectively these results are suggestive of a rapid equilibrium random binding order of Mg2+ and cyclic GMP to the rod phosphodiesterase.