Cultured adult cardiac myocytes are a promising model to study cardiac pathologies on the single cell level. We recently developed a method of long-term culturing (7 days minimum) of adult rat ventricular myoctes with an option for continuous electrical pacing (0.2 and 1Hz). Analysis was performed at day in vitro 0 (freshly isolated), 1, 3 and 6 (DIV0-6). We studied the characteristics of important physiological properties over time i.e. cell length change and intracellular global Ca²⁺ transients during electrical stimulation (0.1-1Hz). Furthermore, we assessed the loading state of internal Ca²⁺ stores with caffeine applications (10mM). Additionally, spatially resolved Ca²⁺ imaging (realtime confocal microscopy) was undertaken to describe the spatiotemporal features of spontaneous Ca²⁺ sparks and waves. Characteristic changes in the functional properties especially at DIV3 and DIV6 were found. Importantly, the continuous electrical pacing at low frequencies did not alter that physiological behaviour. At DIV3 and 6, the reduced twitch amplitudes and Ca²⁺ responses were accompanied by significantly decreased amplitudes of caffeine-induced Ca²⁺ transients: DIV3 (80% decrease; n=5; p<0.001) and DIV6 (29% decrease; n=11; p<0.01). Our work provides a well defined and characterised base for studying the modulation of pathways involved in cardiac pathologies. Fundings: HOMFOR and the DFG: SFB 530, and Graduiertenkolleg "Cellular Regulation and Growth".