Chronic exercise training elicits adaptations in the heart that improve pump function and confer cardioprotection. To identify molecular mechanisms by which exercise training stimulates this favorable phenotype, a proteomic approach was employed to detect rat cardiac proteins that were differentially expressed or modified after exercise training. Exercise-trained rats underwent six weeks of progressive treadmill training five days/week, 0% grade, using an interval training protocol. Sedentary control rats were age- and weight-matched to the exercise-trained rats. Hearts were harvested at various times (0-72 h) after the last bout of exercise and were used to generate 2-D electrophoretic proteome maps and immunoblots. Compared with hearts of sedentary rats, 26 protein spot intensities were significantly altered in hypertrophied hearts of exercise-trained rats (p <0.05), and 12 spots appeared exclusively on gels from hearts of exercise-trained rats. Immunoblotting confirmed that chronic exercise training, but not a single bout of exercise, elicited a 2.5-fold increase in the abundance of one of the candidate proteins in the heart, a 20 kDa heat shock protein (hsp20) that persisted for at least 72 h of detraining. Thus, exercise training alters the cardiac proteome of the rat heart; the changes include a marked increase in the expression of hsp20.