We have investigated the O2-binding properties of the four embryonic hemoglobins (Hb P, Hb P', Hb M, and Hb E) under various conditions and compared the results with measurements on early embryonic chicken blood between 3 and 6 days of incubation. The O2-binding curve of embryonic blood is polyphasic. Hill's coefficient changes continuously from less than or equal to 1 at low O2 saturation to a value of approximately equal to 8 in the upper saturation range. The high cooperativity is coupled with a very low O2 affinity in the middle range of the O2-binding curve. Between 3 and 6 days of development the O2 affinity and cooperativity as well as the Bohr effect of embryonic blood change drastically, without corresponding alterations of the hemoglobin pattern or ATP concentration. The functional properties of the embryonic blood cannot be simulated with the isolated embryonic hemoglobins at physiological concentrations of ATP, hemoglobin, and protons. On the other hand, freshly prepared hemolysate shows the same functional pattern as the embryonic blood. The results suggest that embryonic red blood cells may contain other low-molecular-weight substances that reduce the O2 affinity and increase the cooperativity of the major embryonic hemoglobins, Hb P and Hb P', presumably by promoting tetramer-tetramer aggregation.