Members of the ETS gene family are known to be expressed in hematopoietic tissues and cell lines, and there is increasing evidence that ETS proteins may play a role in normal hematopoietic cell development. We demonstrate that ETS-1 can contribute to the development of an erythroid phenotype in vitro. The pluripotent erythroleukemic K562 and HEL cell lines express messages for a number of ETS genes, but only c-ETS-1 levels are elevated in response to treatment with hemin or cytosine arabinofuranoside (Ara-C), agents which induce erythroid differentiation. Furthermore, ETS-1 antisense oligonucleotides inhibit hemoglobinization of cells treated with Ara-C or hemin, and K562 and HEL cells infected with retrovirus expressing the c-ETS-1 gene exhibit a significant increase in erythroid character (as indicated by benzidine staining for hemoglobin (Hb) and surface marker analysis), a dramatic increase in responsiveness to hemin or Ara-C, and a decreased rate of proliferation (20-40% of control rates). In contrast, infection with virus expressing ETS-2 or vector sequences only causes no detectable changes in the proliferation or erythroid character of either the HEL or K562 cell lines. These data indicate a role for ETS-1 in erythroid differentiation.