A variety of chemical agents have been shown to induce differentiation in in vitro cultured neoplastic cell lines. We noted that blast cells in the peripheral blood of acute nonlymphoid leukemia patients treated with the drug Harringtonine appeared to undergo morphological changes that suggested differentiation. In view of the relatively minimal myelotoxicity of Harringtonine, we hypothesized that harringtonine was acting by differentiation-induction, which concomitantly arrested cell division. We tested this hypothesis using two different experimental approaches. First, the promyelocytic leukemic cell line, HL-60, was cultured with Harringtonine and shown to differentiate into a cell, which, by functional cell surface marker and morphological criteria, closely resembled normal monocytes. Furthermore, these changes were accompanied, and indeed slightly preceded by, loss of proliferative capacity. Second, to prove that the leukemic blasts were the cells undergoing the changes observed in vivo, freshly isolated leukemia cell populations were cultured with Harringtonine, and morphological changes paralleling those seen in the patients were observed. Thus, the antileukemic effect of Harringtonine appeared to be due to diversion of the proliferating blast cells into a differentiation pathway, which, as in normal myeloid cells, resulted in the arrest of proliferation.