To understand the detailed mechanisms of arrhythmia and its treatment methods, it is required to elucidate the mechanisms of formation and debacle of cardiac beat at a microscopic level by using appropriate in vitro models of cardiac beating. In this study, we have developed a method for an medium-exchange-free culture of rat cardiac myocytes, and carried out long-term recording of electrical activity of cardiac myocytes from postnatal rat by means of microelectrode-array (MEA) and the new culture method. Figure 6 a) ∼ f) is a series of the properties of spontaneous beating (mean beating interval, standard deviation and coefficient of variation) of cardiac myocytes cultured by conventional culture method with alternate-day medium exchange (a ∼ c) or newly developed “molecular diffusion culture” method (d ∼ f). As shown in these figures, the difference of the beating property between the individual cultures, which is observed in conventional cultures (a) ∼ c)), was dramatically reduced in the molecular diffusion cultures (d) ∼ f)). In addition, these results also indicated that there is a transient period when all of mean beating interval, standard deviation, and coefficient of variation increase transiently before the formation of stable rhythmic fast beat.