We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I Ca,L), inward rectifier potassium current (IK1), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 ± 70 μm2 on day 0 to 1432 ± 50 μm 2 on day 5), T-tubule density, systolic shortening (from 0.11 ± 0.02 to 0.05 ± 0.01 μm) and amplitude of calcium transients (from 1.54 ± 0.19 to 0.67 ± 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 ± 8 to 105 ± 11 pF) and membrane currents were also reduced (ICa,L, from 3.98 ± 0.39 to 2.12 ± 0.37 pA pF; and IK1, from 34.34p ± 2.31 to 18.00 ± 5.97 pA pF -1). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 ± 2.5 to 34.2 ± 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.