The ability of newborn rabbit hearts to utilize fatty acids as an energy substrate was determined. Isolated working hearts from 1- or 7-day-old rabbits were perfused with Krebs-Henseleit buffer containing either 11 mM glucose or 0.4 mM palmitate as carbon substrates. One-day-old rabbit hearts were perfused at a 11.5-mmHg filling pressure via the inferior vena cava and at a combined aortic and pulmonary arterial hydrostatic afterload of 20 mmHg. In these hearts, addition of insulin was necessary to maintain mechanical function. Function was maintained in the presence of glucose or glucose plus palmitate but not in the presence of palmitate alone. Measurement of glucose and palmitate oxidation rates in hearts perfused with glucose, palmitate, and insulin showed that 57% of ATP production from exogenous substrates was provided by glucose. Substrate use was also measured in 7-day-old rabbit hearts perfused in the Neely working heart mode at a 7.5-mmHg preload and 30-mmHg afterload. In these hearts, function could be maintained in the presence of either glucose alone or palmitate alone. Insulin addition was not necessary to maintain function. Measurement of glucose and palmitate oxidation in 7-day-old rabbit hearts perfused with glucose, palmitate, and insulin showed that only 10% of ATP production from exogenous substrates was provided by glucose. These data demonstrate that between 1 and 7 days of life in the rabbit the heart switches to using predominantly fatty acids as an energy substrate.