Cardiac cells obtained from neonatal rat heart contain a mixed population of cell types that can be enriched in culture in either myocytes or fibroblast-like cells. A metabolic comparison of mixed heart cell cultures with enriched cultures of the same age-in-culture and initial cell density showed that mixed cultures used glucose more rapidly than either enriched myocytes or fibroblasts. Mixed cultures were shown to respond to deprivation of insulin and of serum with decreases in the rate of glucose usage and decreases in the protein content of cells, whereas enriched cultures did not respond in the expected manner to insulin deprivation. Mixed, 11-day-old cells also exhibited greater increases in cellular protein and greater resistance to the stress of starvation than enriched cultures. Palmitate usage, however, was similar in all cultures examined. We conclude that mixed cultures may serve as a better model system to study cardiac metabolism and to monitor the effects of drugs and hormones on the neonatal myocardium. In addition, it is clear from our results that myocytes and fibroblastic-like cells coexist in a metabolically functional synergism.