Revised Manuscript Received September 25, 1989 abstract: We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3, 5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3, 4, 5-trichlorophenylureido) phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al.(1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, andthe pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 Á from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill’s coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made toinduce the same effects as L345 alone by adding inositol hexaphosphate. Both compoundsincrease the alkaline and acid Bohr effects. Theyalter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reactingfraction inthe R state. When [L345]> 1