N2-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O2 followed by N2 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 O2 perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T1), and spin-spin relaxation time (T2), under perfusion with N2 or Ar as opposed to O2, indicated that loss of signal was due to T1 saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T1, values derived from biexponential relaxation processes under perfusing O2 were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N2 these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T1 relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T2 values for cortical tissue were also biexponential but were essentially the same under O2 (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N2 (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 T1 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 T1 experiments demonstrate that O2 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 O2 perfusion) is responsible for the observed relaxation changes.