The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5' untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum-responsive eIF4B phosphorylation site, Ser422, located in an RNA-binding region required for eIF4A helicase-promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide-3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin-resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.