The purpose of this study was to try to reconstitute three-dimensional cardiac tissue using a thermoresponsive artificial extracellular matrix, poly (N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin), as the scaffold. PNIPAM-gelatin solution gels almost immediately when heated above 34°C. We thought this property could become advantageous as scaffolding for reconstituting three-dimensional tissue. Because PNIPAM-gelatin solution gels so quickly, all seeded cells in PNIPAM-gelatin solution would become entrapped and uniformly distributed toward three dimensions. Thus it would be possible to reconstitute three-dimensional tissue by a very simple method of mixing cells and PNIPAM-gelatin solution. Fetal rat cardiac cells were mixed with PNIPAM-gelatin solution, incubated at 37°C to allow the mixture to gel, and cultured in vitro. To define suitable culture conditions the following parameters were tested: (1) PNIPAM-gelatin concentration, 0.04±0.125 mg/ml; (2) cell seeding density, 1~50 × 106 cells/ml; and (3) addition or not of hyaluronic acid. With a PNIPAM-gelatin concentration of 0.05 mg/ml, a cell seeding density of 50 × 106 cells/ml, and the addition of hyaluronic acid, tissue was reconstituted and it contracted synchronously. After hematoxylin and eosin staining, the cells reconstituted three-dimensional tissue, and the tissue cross-section was approximately 60 μm thick.