Cardiac tissue engineering aims to create myocardial patches for repair of defective or damaged native heart muscle. The inclusion of non-myocytes in engineered cardiac tissues has been shown to improve the properties of cardiac tissue compared to tissues engineered from enriched populations of myocytes alone. While attempts have been made to mix non-myocytes (fibroblasts, endothelial cells) with cardiomyocytes, very little is understood about how the tissue properties are affected by varying the respective ratios of the three cell types and how these cells assemble into functional tissues with time. The goal of this study was to investigate the effects of modulating the ratios of the three cell types and to spatially and temporally track cardiac tri-cultures of cells. Primary neonatal cardiac fibroblasts and D4T endothelial cells were incubated in 5 mu m CellTracker (TM) green dye and CellTracker (TM) red dye, respectively, while neonatal cardiomyocytes were labelled with 20 mu g/ml, DAPI. The non-myocytes were seeded either sequentially (pre-culture) or simultaneously (tri-culture) in Matrigel-coated microchannels and allowed to form organoids, as in our previous studies. We also varied the seeding percentage of cardiomyocytes while keeping the total cell number constant in an attempt to improve the functional properties of the organoids. Organoids were imaged on days 1 and 4. Endothelial cells were seen to aggregate into clusters when simultaneously tri-cultured with myocytes and fibroblasts, while pre-cultures contained elongated cells. Functional properties of organoids were improved by increasing the seeding percentage of enriched cardiomyocytes from 40% to 80%. Copyright (c) 2009 John Wiley & Sons, Ltd.