A survey of phosphorus compounds present in HeLa cells and their acid extracts has been carried out by (31)P nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled (31)P spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 mumol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD(+), glucose 6-phosphate, beta-D-fructose 1,6-bisphosphate, alpha-D-fructose 1,6-bisphosphate, ADP, alpha-glycerophosphorylcholine, and alpha-glycerophosphorylethanolamine. UTP made up (1/5) of the total nucleotide triphosphate content. The composition was compared to the (31)P spectrum of an extract from a human astrocytoma grown in athymic mice. The signal from P-containing macromolecules such as nucleic acids was not detected in the intact HeLa cell spectrum because of broad lines. Effects of the glycolysis inhibitor iodoacetic acid could be clearly shown in spectra of both the intact cell and the extract as buildup of fructose 1,6-bisphosphate at the expense of ATP, UTP, and creatine phosphate.