Human embryonic ζ and ɛ globin chains are synthesized in yolk sac–derived primitive erythroid cells, and decrease rapidly during definitive erythropoiesis. Examination of ζ and ɛ globin expression at the cellular level using dual-color immunofluorescence staining with specific monoclonal antibodies showed that embryonic globin proteins are present in definitive erythroid cells. More than half of fetal erythrocytes were positive for ζ and ∼5% for ɛ globin. Approximately one third of newborn red blood cells were ζ-positive and less than 1% ɛ-positive. Adult erythrocytes did not have embryonic globins. Erythroblasts that developed in liquid cultures also contained embryonic globin in amounts which declined with ontogenic age, and the proportion of positive cells in vitro was less than in the comparable erythrocytes that developed in vivo. Thus, embryonic globin chains are synthesized in definitive erythroid cells and decrease with ontogeny. Modulation of embryonic globin gene expression is not solely due to a switch from primitive to definitive erythropoiesis.