The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb-O2 equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory. At low concentrations (NTP/Hb ratio = 1.0, and pH greater than 7.3) ATP, GTP and protons decrease Hb-O2 affinity by increasing the allosteric constant L and reducing KT, the association constant of the deoxy, tense state of the Hb, without significantly affecting that (KR) of the oxy state, increasing the free energy of cooperativity (delta G). High concentrations of these effectors, however, also reduce KR. The greater sensitivity of the half-saturation O2 tension (P50) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on both KT and KR. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the T----R transition basic to cooperativity in fish Hb. The possible implications of the findings for aquatic respiration are discussed.