Abstract Addition of fresh medium to stationary cells of Arabidopsis suspension culture induces increased phosphorylation of the S6 ribosomal protein and activation of its cognate kinase, AtS6k. Analysis of the activation response revealed that medium constituents required for S6 kinase activation were the phytohormones 1-naphthylacetic acid (auxin) and kinetin. Pretreatment of cells with anti-auxin or PI3-kinase drugs inhibited this response. Consistent with these findings, LY294002, a PI3-kinase inhibitor, efficiently suppressed phytohormone-induced S6 phosphorylation and translational up-regulation of ribosomal protein S6 and S18A mRNAs without affecting global translation. These data indicate that (1) activation of AtS6k is regulated by phytohormones, at least in part, via a lipid kinase-dependent pathway, that (2) the translational regulation of ribosomal proteins appears to be conserved throughout the plant and animal kingdom, and that (3) these events are hallmarks of a growth-related signal transduction pathway novel in plants.