The amino acids and tryptic peptides that become phosphorylated in 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells were examined by two-dimensional thin-layer electrophoresis. In the maximally phosphorylated form of the protein, most of the phosphate was incorporated into serine and a small amount, into threonine. Digestion of this form of the protein with trypsin revealed 10 major phosphopeptides. All 10 contained phosphoserine and 2 of the 10 also contained phosphothreonine. Next, the five forms of increasingly phosphorylated S6 were individually separated on two-dimensional polyacrylamide gels or total S6 was isolated from cells that were stimulated for only a short time and their phosphotryptic maps were analyzed. The results showed that, as larger amounts of phosphate were added to S6, the phosphopeptides appeared in a specific order.