To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi- 1 leukemic cell lines, the last from a t(8;21) AML. Biochemical analysis confirmed specificity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipitations of metabolically labeled 32P- proteins from Kasumi-1 cells show that AML1 and ETO are phosphorylated on serine and threonine. Investigations with normal bone marrow reveal AML1 and ETO are coexpressed in megakaryocytes and that each is expressed in a portion of the approximately 10-microns-diameter cells residing there. Using a CD34+ enriched population mobilized to peripheral blood, we found AML1 and, unexpectedly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic. Also, because ETO would not be exclusively expressed as part of chimeric AML1/ETO in leukemic patients, its presence cannot be used to monitor t(8;21) AML residual disease.