Previous experiments using cross-linked tetrameric hemoglobins (XLHb) to perfuse isolated rat kidneys showed that high O₂-affinity XLHb improved proximal tubule function more effectively than low O₂-affinity XLHb. To determine how function was improved, proximal tubule fragments were incubated with albumin, Hb₃₄ (P₅₀ 34 mm Hg) or Hb₁₃ (P₅₀ 13 mm Hg) with pO₂s ranging from 22-147 mm Hg. ATP content reflected O₂ delivery to mitochondria. Both XLHbs increased ATP; Hb₃₄ with pO₂ 47 mm Hg and Hb₁₃ with pO₂ 47 mm Hg. XLHbs increased NaKATPase activity ( ⁸⁶Rb uptake) in similar pO₂-dependent patterns. O₂-consumption (QO₂) was measured in a closed well-stirred chamber. Ouabain- and oligomycin-inhibited QO₂, reflecting NaKATPase activity and oxidative phosphorylation respectively, mirrored the pO₂ dependent patterns of ATP and ⁸⁶Rb uptake. As pO₂ fell below the mid-point of XLHb desaturation, QO₂, uncoupled from oxidative phosphorylation, transiently increased. The increase was most pronounced with Hb₃₄. L-NAME had no effect on QO₂. Inhibitors of NAD(P)H oxidases and diamine oxidase partially prevented the QO₂ surge with Hb₃₄. Conclusions. Facilitated diffusion accounts for pO₂-dependent XLHb effects on ATP content and NaKATPase and for Hb₁₃'s effectiveness in hypoxic perfused kidneys. NO scavenging was not a factor. O₂-binding characteristics influence XLHb effects on mitochondria and O₂-sensitive enzymes such as oxidases.