31P nuclear magnetic resonance spectra of th phototrophic bacterium Rhodopseudomonas sphaeroides reveal the presence of inorganic phosphate, sugar phosphates and two non-identified P,P1-diesterified pyrophosphate compounds. Due to the presence of paramagnetic cations the resonances of these compounds can only be detected after repeated washing of the bacterial cells with a buffer, containing EDTA plus excess Mg2+. Washing with Mg2+-free EDTA buffer deteriorates the structural integrity of the membranes of Rps. sphaeroides. This is indicated by the appearance of an extra resonance peak in the spectra of these cells in a region where the phospholipids absorb and by a fivefold increase in proton permeability of the cytoplasmic membrane of Rps. sphaeroides under these conditions. Upon illumination of the cell suspension in the NMR tube the generation of a transmembrane pH gradient can be inferred from the shift in the resonances of extracellular and intracellular inorganic phosphate. Intracellular inorganic phosphate shows one homogeneous resonance peak upon illumination. This demonstrates that the mixing system, which has been developed for this application, functions efficiently. The magnitude of the light-dependent pH difference is 0.8 at the external pH 6. The width at half height of the internal inorganic phosphate peak is essentially independent of internal pH from pH 5--8, remains unchanged upon addition of uncoupler and is inversely proportional to the number of EDTA washings applied. These observations indicate that the inorganic phosphate NMR peak width is predominantly determined by the presence of a residual amount of paramagnetic cations, rather than by a broad distribution of internal pH values over the cells. Ionophores have an effect on the light-dependent pH-gradient in accordance with the chemiosmotic theory: valinomycin increases, and carbonylcyanide p-trifluoromethoxyphenylhydrazone decreases, the magnitude of this gradient.