Cardiac myocytes isolated from adult rat ventricles have been maintained in a stable, differentiated state for prolonged periods by the use of suspension culture on hydrophobic tissue culture inserts or agarose-coated plates. The success of this procedure depends on the use of low-serum media to prevent myocyte-myocyte interaction and proliferation of any residual endothelial cells. Myocytes cultured in this manner retain many of their structural characteristics, suggesting that maintenance of their elongated irregular shape is not dependent on interaction with extracellular matrix. They also exclude trypan blue, can be vitally stained by the uptake and reduction of the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], synthesize myosin and when returned to adhesive surfaces are capable of attachment and attendant dedifferentiation. Stability of the myocytes in suspension permits their use in co-culture experiments; specifically, myocytes separated from endothelial cells by the hydrophobic membrane of the tissue culture insert stimulated proliferation of the latter cells, suggesting this to be a useful system for studying myocyte-endothelial cell interaction.