The mechanism, by which aldosterone causes cardiac fibrosis is not well understood. Therefore, additional effects have been suggested for aldosterone-mediated fibrosis. In this study, we investigated the effect of aldosterone in combination with a second agonist on fibrotic mechanisms using primary cultures of fibroblasts from adult rat hearts. The cultured fibroblasts were identified by labelling of vimentin and collagen receptor 2. These cultures did not show a-smooth muscle actin-positive cells (<10%). Mineralocorticoid receptor is expressed in cardiac fibroblasts analysed by RT-PCR. Aldosterone (10 nM) alone did not change protein amount, proliferation or concentration of the extracellular matrix compounds (collagen I, III, IV or fibronectin) and did not cause apoptotic or necrotic cell death. Angiotenin II (100 nM) increased the protein amount but reduced apoptosis and the extracellular matrix compounds (collagen I, III and fibronectin). These effects were not altered by aldosterone. The interaction of aldosterone and isoprenaline (1 mM) induced apoptosis comparable to the isoprenaline effect. The analysis of the extracellular matrix compounds showed that both aldosterone and isoprenaline were necessary to increase collagen IV. Whereas the isoprenaline-dependent induction of fibronectin was blocked by aldosterone. An enhanced salt-concentration (plus 15 mM Na+) induced apoptosis. Addition of aldosterone during an enhanced salt level reduced the amount of collagen III. Whereas the salt-caused reduction of collagen IV was blocked by aldosterone. The interaction of aldosterone and an endogenous cardiac glycoside (1 mM marinobufagenin) caused apoptosis but showed no effect on the extracellular matrix compounds. None of the combinations of aldosterone with a second agonist changed proliferation. Besides, necrosis determined by the relative LDH activity was not enhanced by the investigated combinations with aldosterone. In conclusion our results indicate that the aldosterone-mediated fibrosis involves more than a second stimulus and proves to be a complex process.