Two protein kinases differing in substrate specificity were used to phosphorylate the 30-S and the 50-S ribosomal subunits of Escherichia coli. The catalytic subunit from the rabbit skeletal muscle protein kinase phosphorylates proteins S1, S4, S9, S13 and S18 of the 30-S subunit and proteins L2, L4, L5, L16, L18 and L23 of the 50-S subunit with (gamma-32P)ATP as phosphoryl donor. A second protein kinase isolated from rabbit reticulocytes, formerly shown to phosphorylate preferentially acidic proteins and to use GTP as well as ATP, strongly phosphorylated protein S6, an acidic protein of the small ribosomal subunit, and to a lesser extent proteins L7 and L12 or the large subunit. Evidence is presented showing different phosphorylation patterns when either whole subunits or the extracted proteins were used as substrate for the protein kinase. Kinetic studies showed proteins S1 and S4 to become most rapidly phosphorylated. Although most proteins incorporated less than stoichiometric amounts of phosphate, it is shown that with a high excess of ATP L2 bound 1 mol phosphate/mol protein.