When tested in a poly(U)-dependent polyphenylalanine synthesizing system and in a postnuclear supernatant, both derived from Ehrlich ascites tumor cells, 2′(3′),5′-ADP did not affect chain elongation of polypeptide synthesis. In a cell-free system which was dependent on initiation and programmed by natural mRNA, however, the amino acid incorporating activity was suppressed to about 10% of the control in the presence of 1 mM 2′(3′),5′-ADP. The inhibitor was shown not to interfere with the attachment of poly(U) to the small ribosomal subunit and with the formation of mRNA-80S ribosome complexes in a complete protein synthesizing system. The subsequent attachment of a 40S ribosomal subunit to the mRNA-80S ribosome complex and the formation of polysomes, however, was depressed by the inhibitor. The experimental results suggest that 2′(3′),5′-ADP inhibits initiation-dependent protein synthesis between monosome formation and the formation of the first peptide bond(s).