Of the two major intracellular divalent cations, Ca2+ has been studied much more extensively than Mg2+ and is now well accepted as a major intracellular regulator. This review focuses instead on some recent advances in the understanding of the physiology and biochemistry of Mg2+. For purposes of discussion, four criteria have been developed that should be fulfilled if Mg2+ is to be accepted as an important intracellular regulatory cation: cellular processes must exist which are sensitive to free Mg2+ within the physiological concentration range; a (transport) mechanism(s) must exist which is capable of altering free Mg2+ concentration within a cell; if Mg2+ is compartmented within cells, any potentially regulated system or process and any change in intracellular free Mg2+ concentration must be shown to occur within the same compartment; and any change(s) in free Mg2+ concentration and any alteration(s) in a Mg2+-sensitive process must occur in a sequential manner. These criteria are largely but not completely met at the present time. Criteria 1 and probably 2 can be shown in at least some systems to be fully met. Criteria 3 and 4 are partially met but neither can be fully examined until methods for measuring intracellular free Mg2+ concentrations on an appropriate time scale are further developed. Thus, there exists strong but as yet incomplete evidence that Mg2+, like Ca2+, can play an active, regulatory role within cells. Finally, it is suggested that Ca2+ plays the specific role of the acute, transient regulatory element while Mg2+ plays the complementary role of a more long-term regulatory element which controls the set point or gain of a system or process.