The Cephalotaxus alkaloid harringtonine inhibits protein biosynthesis in HeLa cells, intact rabbit reticulocytes, and reticulocyte lysates. DNA synthesis in HeLa cells is partially inhibited by a 200 nM concentration of the alkaloid, while synthesis of RNA is unaffected; the inhibitory effects on protein and DNA synthesis are partially reversible. Harringtonine induces breakdown of polyribosomes to monosomes with concomitant release of completed globin chains. When the alkaloid is added to a reticulocyte lysate, a delay of 2 min occurs before inhibition of globin synthesis is observed. Based on the preceding observations, the principal effect of harringtonine appears to be on initiation of protein synthesis. In contrast to some other inhibitors of initiation, harringtonine does not inhibit binding of polyuridylic acid or tRNA to ribosomes, nor does it inhibit chain elongation even at high concentrations of drug. These observations provide a biochemical basis for the cytotoxic and chemotherapeutic properties of harringtonine and suggest its use as a tool for the study of protein synthesis in animal cells.