1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.