The mitogen-activated 70K S6 kinase has an apparent Km for 40 S ribosomal subunits of 0.25 microM. The apparent Km for a synthetic peptide derived from the carboxyl terminus of S6 and containing all of the in vivo sites of phosphorylation was 2.5-fold higher. A number of shorter peptides revealed that the substrate recognition determinants for the preferred site of phosphorylation, Ser236, reside in a seven-amino acid stretch of S6, residues 231-217. Critical to recognition is a block of 3 consecutive arginines, especially Arg231 and Arg233. In contrast, replacement of Ser235 or the preferred site of phosphorylation, Ser236, with alanine has little effect on the apparent Km. Based on this data the consensus recognition sequence would be Arg-(Arg)-Arg-X-X-Ser-X. A number of kinases known to phosphorylate S6, including cAMP-dependent protein kinase and protein kinase C and the 92K S6 kinase II, were also tested for their ability to phosphorylate a decapeptide containing all the critical recognition determinants. Finally, a synthetic peptide containing a putative 70K S6 kinase autoinhibitory domain did not serve as a substrate for the enzyme but did inhibit its activity, although much less effectively than a synthetic peptide containing all the recognition determinants.