To investigate the roles that the community effect and entrainment function of cultured cardiomyocyte play in decreasing beating fluctuation and reestablishing synchronized beating, we developed a single-cell-based two-dimensional network culture assay to measure and compare the dynamics of beating rhythm synchronization of individual cells before and after they form networks. Studying the formation of two-cell networks, we found that their synchronized beating tended to be determined by the cardiomyocyte whose beat rate fluctuated less than that of the other cardiomyocyte. We further found that the strength of this tendency increased with the number of cells in the network. These results indicate that (1) beating fluctuation is one of the important factors influencing the reestablishment of a stable synchronous beating rhythm, (2) the larger networks reduce fluctuation, and (3) the formation of a spatial network can itself stabilize cardiomyocyte beat rates. (c) 2006 Elsevier Inc. All rights reserved.