Heart cells cultured in vitro in primary cell culture are useful in the investigation of the differentiation of the heart on the cellular level. Atrial and ventricular cells from embryonic chick hearts and ventricular cells from the hearts of 1- to 2-day old rats may be cultured in the chemically defined nutrient media SM 20 and SM 20-I. These media are prepared without lipids or fatty acids. Neither do they contain proteins or other macromolecules, or undefined substances of smaller size. Pulsating high density cell layers may be cultured in SM 20 for 20 to 30 days and in SM 20-I for 15 to 20 days. The cells exhibit a certain degree of functional and structural differentiation during these periods of cultivation. There is no evidence that they proliferate during this time. It is not yet known to what extent the differentiation of heart muscle cells may be influenced by the presence of other cell types in a culture or by constituents or products of surrounding cells.