The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.