Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of α and β subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of α and β hemes separately. Previous studies have characterized the oxygen binding by α(Ni2+)2 β(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of α(Fe2+)2 β(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 ± 3 and 87 ± 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 ± 0.06 and 0.90 ± 0.03. Analysis of the binding curves, taking into account the different projections of the α hemes along the optical directions, indicates that the oxygen affinity of α1 hemes is 1.3-fold lower than α2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the β subunits of α(Ni2+)2 β(Fe2+)2 crystals. The average oxygen affinity of the α subunits (p50 = 91 Torr) is about 1.2-fold higher than the β subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.