Eukaryotic initiation factors (eIF)-4A, -4B, and -4F isolated from wheat germ were tested for their ability to catalyze ATP hydrolysis in the absence and presence of RNA. We find that eIF-4B or eIF-4F alone exhibit ATPase activity in the presence of poly(U), satellite tobacco necrosis virus (STNV) RNA, or globin mRNA but not in the absence of RNA. eIF-4A alone exhibits ATPase activity in the absence of RNA, but this activity is not stimulated by the addition of RNA. eIF-4A does, however, enhance RNA-dependent ATP hydrolysis in the presence of either eIF-4B or eIF-4F. The RNA-dependent ATPase activities of eIF-4B and eIF-4F are additive, not synergistic. At saturating concentrations of eIF-4F, little or no stimulation of ATP hydrolysis is obtained upon the addition of eIF-4B and at saturating concentrations of eIF-4B little or no stimulation is obtained upon the addition of eIF-4F. This observation is in agreement with our previous finding (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 330-333) that initiation of polypeptide synthesis is obtained in vitro with either eIF-4F or eIF-4B.