Creatine kinase from the smooth muscle of the cow uterus was extracted and purified by procedures involving precipitation of the enzyme in the presence of ethanol, cation exchange chromatography on phosphocellulose, gel filtration in Sephadex G-150 and anion exchange chromatography on DEAE-cellulose. The purified enzyme eluted as a single active peak after rechromatography on Sephadex G-150 with a molecular weight of about 82 000. Electrophoresis in polyacrylamide gels in tris-glycine buffer (pH 8.6) under non-denaturing conditions revealed a single enzymatically active protein band. In the presence of sodium dodecyl sulphate, the enzyme migrated as a single band in polyacrylamide gels at a mobility corresponding to a molecular weight of about 40 000 per subunit. Reaction with iodoacetamide indicated the presence of sulphydryl groups of differing susceptibility to alkylation. The purified enzyme was optimally active over a wide pH range (6.5-8.0). The Michaelis constants (Km) of the enzyme for MgADP and phosphoryl creatine (PCr) are 0.12 mM and 0.7 mM respectively, which are significantly lower than those for skeletal muscle CK. MgADP lowered the dissociation constant of the enzyme for PCr (from about 3.6 mM to 0.7 mM). Evidence is presented that the high affinity of the smooth muscle CK to MgADP and the MgADP-mediated facilitation of PCr binding might be key factors in the role of this enzyme in harnessing the energy reserves of the cell.