Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.