31P nuclear magnetic resonance spectroscopy has been used to study freshly aspirated normal human bone marrow samples. The pH within the intact cells of the samples was determined from the chemical-shift position of the resonance for inorganic phosphate within the cell; the intracellular pH was found to be 7.35 for fresh bone marrow. The chemical-shift positions of the alpha and beta phosphate resonances of adenosine 5'-triphosphate were used to assess the fraction of this metabolite complexed with Mg2+. It was found that 84% of the total intracellular adenosine 5'-triphosphate was in the Mg2+-complexed form. The concentration of Mg2+ uncomplexed to any ligand was 0.4 mM. The areas of the resonances for the major phosphorus-containing metabolites were used to determine intracellular concentrations. For fresh human bone marrow, the intracellular concentrations determined were phosphate monoesters less than 0.3 mM, 2,3-diphosphoglycerate 3.9 +/- 1.0 mM, inorganic phosphate 1.2 +/- 0.6 mM, phosphodiesters 2.8 +/- 1.0 mM, adenosine 5'-triphosphate 1.6 +/- 0.4 mM, adenosine 5-diphosphate less than 0.2 mM, and nicotinamide adenine dinucleotide less than 0.2 mM. These metabolite concentrations within the intact cell samples did not change over 2.0 h and changed only gradually over a 24-h period. 31P nuclear magnetic resonance spectroscopy was then used to study the cryopreservation of normal human bone marrow in the presence of increasing concentrations of the penetrating cryopreservative dimethyl sulfoxide. Dimethyl sulfoxide alone without freezing was found to cause some gradual hydrolysis of 2,3-diphosphoglycerate, presumably by stimulating diphosphoglycerate phosphatase. The effect of freezing human bone marrow to liquid nitrogen temperatures, storage, and rapid thawing was a dramatic fall in intracellular adenosine 5'-triphosphate levels. The half-life of the metabolite, after thawing, was about 0.3 h. If the bone marrow was frozen in the presence of 2.5, 5.0, and 7.5% dimethyl sulfoxide, the post-thaw half-life was prolonged to 0.4, 0.5, and 0.6 h, respectively. 15% dimethyl sulfoxide afforded complete cryoprotection, with adenosine 5'-triphosphate levels constant for 15 h after thawing the human bone marrow.