We hypothesized that functional constructs with physiologic cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation, and characterized in detail construct metabolism, structure and function. Neonatal rat cardiomyocytes suspended in Matrigel® were cultured on collagen sponges at high initial density (1.35^.10⁸ cells/cm³) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity and cell cycle, presence of contractile proteins (sarcomeric -actin, troponin I and tropomyosin) and contractile function in response to electrical stimulation (excitation threshold, ET, maximum capture rate, MCR, response to a gap junctional blocker). Interstitial flow of culture medium through the central 5 mm diameter x 1.5 mm thick region resulted in physiologic density of viable and differentiated, aerobically metabolizing, cells, whereas dish culture resulted in constructs with only a 100 - 200 µm thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase as compared to dish grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR following treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.