Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these “orthogonal” methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.