Cardiotoxicity studies using isolated heart cells are becoming increasingly advocated as a supplement to, and sometimes as a replacement for, whole heart or whole animal experimentation. In fact, the use of isolated cardiomyocytes has the great advantage of enabling mechanistic and comparative studies of compounds, which are directly toxic to cardiomyocytes. Since the 1970s, different procedures have been developed in order to obtain Ca(2+)-tolerant cardiomyocytes. The advances in this field will be reviewed and an optimised method to obtain freshly isolated Ca(2+)-tolerant cardiomyocytes from the adult rat for use in toxicological studies will be described. With this procedure, a high number of rod-shaped cells can be obtained (6-7 x 10(6)/heart corresponding to 70% of total number of cells). It is also possible to maintain cell viability, glutathione content and enzymatic activity of glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione S-transferase (GST) in acceptable levels for 4 hours. Cardiotoxicity studies performed with isoproterenol (ISO) in the presence of copper and with the model toxic substance tert-butylhydroperoxide (t-BHP) demonstrate the importance of oxidative stress as a cardiotoxic mechanism elicited by these molecules. The results obtained are also good indicators for future applications of this methodology to other cardiotoxicity studies.