Myosin has been isolated from baby hamster kidney cells (BHK21/C13) in high yield and characterized biochemically and immunologically. The subunit composition consists of 2 heavy chains, approximately 200,000 Daltons each, and 2 classes of light chains of approximately 16,000 and 20,000 Daltons. The myosin exhibits ATPase activity in the presence of K+-EDTA or Ca2+ but very little activity with Mg2+-ATP. The Mg2+-ATPase activity is stimulated only about 2-fold by skeletal actin, but a much larger activation is obtained in the presence of a protein kinase isolated from chicken gizzard. The increase in actin activation is accompained by the phosphorylation of the 20,000-Dalton light chain. BHK21 myosin is insoluble at low ionic strength and forms typical biopolar thick filaments. A specific antiserum generated against this protein forms a single precipitin line with the antigen but does not crossreact with either skeletal or smooth muscle myosin. The antiserum also specifically stains stress fibres in BHK21 cells as shown by indirect immunofluorescence.