A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.