1. Physiological concentrations of either Ca2+ or Mg2+ stimulated L-glycerol 3-phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. L-glycerol-3-phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations. 2. Substrate activation of mitochondrial L-glycerol-3-phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate-binding site functioning with high turnover. 3. Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies. 4. From the solubilized mitochondrial proteins L-glycerol-3-phosphate dehydrogenase was bound selectively to the hydrophobic phenyl-Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.