¹³C- and ³¹P-NMR were used in methylene blue-treated human erythrocytes to determine the dependence on intracellular Mg²⁺ concentration ([Mg²⁺]_i) of the pentose phosphate pathway (PPP), the glycolytic pathway, and adenine nucleotide synthesis. The PPP flux had an [Mg²⁺]_i at half-maximal velocity ([Mg²⁺]_i,0.5) of 0.02 mM, well below the physiological range (0.2-0.7 mM). Flux through the PPP was reduced at higher [Mg²⁺]_i as flux through phosphofructokinase was increased ([Mg²⁺]_i,0.5 = 0.16 mM). [Mg²⁺]_i,0.5 of phosphoglycerate kinase flux, which equals net ADP phosphorylation rate, was 0.27 mM, well within the physiological [Mg²⁺]_i range. The rate of adenine nucleotide synthesis from [2-¹³C]glucose-derived ribose 5-phosphate and exogenous adenine also exhibited dependence on [Mg²⁺]_i but was not saturable up to 1.6 mM. Therefore, net flux through the PPP and glycolytic pathways in erythrocytes is not strongly dependent on [Mg²⁺]_i at physiological ion concentrations, but both ADP phosphorylation and adenine nucleotide synthesis are likely to be regulated by normal fluctuations in [Mg²⁺]_i.