We characterized the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analyzed their capacity to phosphorylate the myogenic factor SRF. Isoforms of CaMKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoichiometries and produced multiple forms on SDS-PAGE, suggesting that SRF was phosphorylated at multiple sites. Phosphopeptide-mapping experiments using truncated SRF proteins located the residues of SRF phosphorylated by recombinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides identified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SRF, were sites of phosphorylation. CaMKII activity was enriched in nuclear extracts relative to crude homogenates from skeletal muscle and similarly phosphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by nuclear CaMKII may directly influence DNA binding of SRF and other MADS box factors.