Squamate hemoglobins are responsive to modulation by nucleoside triphosphates (NTP, generally ATP), and bind NTP with a 1-to-1 molar stoichiometry. However, red cells of nonpregnant rattlesnakes contain NTP-to-hemoglobin molar ratios of approximately 2.5 suggesting most NTP is supersaturating and should not influence the oxygen affinity directly. To test this hypothesis, we metabolically depleted red cells of NTP and determined the oxygen affinity. There was a significant linear relationship between red cell NTP concentrations and oxygen affinity over the NTP/Hb range examined. In contrast, intracellular pH, Mg2+ and C1- changed slightly, or not all, during depletion. These data indicate NTP concentrations represent the primary control of hemoglobin function within these cells. Purified hemoglobin was functionally sensitive to 5 mM GTP or inositol hexaphosphate but not sensitive to 5 mM ATP or pH. Together, these findings indicate rattlesnake hemoglobin, within red cells, is functionally controlled by NTP, but the binding affinity is low such that NTP is not saturating at NTP/Hb ratios below 3.5.