A caldesmon kinase activity was partially purified from an extract of chicken gizzard smooth muscle by sequential chromatography on columns of DEAE-Sephacel, MonoQ and Superose 12. This kinase was identified as casein kinase II by Western blotting using peptide-directed antibodies raised against the alpha, alpha' and beta subunits of human casein kinase II; the smooth muscle enzyme consisted of similar subunits of M(r) 43,000 (alpha), 39,000 (alpha'), and 27,000 (beta). Phosphorylation of caldesmon and casein by smooth muscle casein kinase II was optimal at approximately 0.1 M NaCl, did not require second messengers, and was inhibited by heparin. The kinase utilized either GTP or ATP as a substrate. Caldesmon was phosphorylated to approximately 1 mol Pi mol-1 caldesmon by smooth muscle casein kinase II with a Km for caldesmon of 4.9 microM. Two-dimensional thin-layer electrophoresis indicated phosphate incorporation into both serine and threonine. All the incorporated phosphate was recovered in the N-terminal peptide (residues 1-152) generated by cleavage at cysteine 153 with 2-nitro-5-thiocyanobenzoic acid. Purification of tryptic phosphopeptides and N-terminal sequencing revealed two principal sites of phosphorylation: serine 73 and threonine 83. The following four synthetic peptides corresponding to this domain of caldesmon were examined as substrates of casein kinase II: A = RRREVNAQNSVAEEE; B = AQNSVAEEE; C = RSTDDEAA; D = SVAEEETKRSTDDE. Interestingly, only peptides C and D were phosphorylated and both only at threonine. Phosphorylation of intact caldesmon did not affect the pattern of chymotryptic digestion suggesting that it does not induce a significant conformational change in the protein substrate. Phosphorylation also had no effect on the binding of caldesmon to actin or on the caldesmon-mediated inhibition of actomyosin MgATPase activity. However, phosphorylation completely abolished the interaction of caldesmon with immobilized smooth muscle myosin. These results are consistent with the localization of the myosin-binding domain near the N-terminus of caldesmon and of the actin-binding domain near the opposite end of the elongated molecule. Casein kinase II may therefore play a role in regulating caldesmon-myosin interaction and the ability of caldesmon to cross-link actin and myosin filaments in smooth muscle.