Heart component influencing the maintenances of spreading and beating of rat myocardial cells in serum-free culture was investigated. Spreading and well-spreading % of single cells cultured with sample in Eagle minimum essential medium (MEM)-0.5% bovine serum albumin for 2 days at 37°, and relative beating % and rate of single well-spreading cells incubated with sample in Eagle MEM for 1 hr at 37° were used as parameters of the maintenance activities. Culture filtrates of heart ventricle fragments cultured in Eagle MEM for 4 days at 37° significantly promoted all of 4 parameters, and precipitates salted out with 30-50% saturated ammonium sulfate from aqueous alkaline extracts of rat, bovine and rabbit ventricles had the promoting effects like culture filtrate. But extracts prepared from brain, liver, spleen, pancreas, small intestine, kidney and skeletal muscle in the same manner as done in ventricles did not affect or inhibited spreading and beating. Desalted culture filtrates and extracts of ventricles did not possess trypsin inhibitory activity. Bovine ventricle extract was fractionated for an active principle using successive chromatographies on DEAE-cellulose, Sephadex G-100 and CM-cellulose column. The isolated principle, Fr. BVP (bovine ventricle protein), was a protein possessing molecular weight of 100000 and 18 kinds of amino acids. Fr. BVP significantly maintained spreading and beating of myocardial cells in serum-free culture in a concentration of 100 μg/dish. This protein differed biologically and chemically from Fr. A, inotropic protein of bovine heart, which was effective for promoting the beating behaviors of myocardial cells cultured with serum.