N(2)-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O(2) followed by N(2) or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O(2) perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T(1)), and spin-spin relaxation time (T(2)), under perfusion with N(2) or Ar as opposed to O(2), indicated that loss of signal was due to T(1) saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T(1), values derived from biexponential relaxation processes under perfusing O(2) were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N(2) these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T(1) relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T(2) values for cortical tissue were also biexponential but were essentially the same under O(2) (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N(2) (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T(1) decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T(1) experiments demonstrate that O(2) does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O(2) perfusion) is responsible for the observed relaxation changes.