Human blood was diluted in isotonic saline at pH 7.4 and deoxygenated. After gentle exposure to nitric oxide gas (NO), the red blood cells (erythrocytes) remained intact. Increasing the cell volume fraction allowed detection of strong electron paramagnetic (EPR) signals, even at ambient temperature (293 K). These spectra were compared to those recorded at 77 K. With maximal NO exposure, a relatively featureless and stable spectrum was recorded. Reduced exposure produced a spectrum, which gradually transformed into the final one, with more structure. The spectral features with unfrozen samples reflected the degree of resolution of the hyperfine triplet component observed at 77 K. This hyperfine coupling was independent of the temperature. The spectra reveal rearrangement of the NO ligand between the subunits of hemoglobin. At subsaturation levels, binding to the alpha subunit, with its iron penta-coordinated in the T-state, dominates. (C) 1994 Academic Press, Inc.