Mechanical load and chemical factors as stimuli for the different pattern of the extracellular matrix (ECM) could be responsible for cardiac dysfunction. Since fibroblasts can both synthesize and degrade ECM, ventricular fibroblasts from adult rat hearts underwent cyclical mechanical stretch (CMS; 0.33 Hz) by three different elongations (3%, 6%, 9%) and four different serum concentrations (0%, 0.5%, 5%, 10%) within 24 h. Expression of collagen I and III, as well as matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and colligin were analyzed by RNase protection assay. In the absence of serum, 9% CMS increased the mRNA of collagen I by 1.70-fold and collagen III by 1.64-fold. This increase was prevented by the inhibition either of PKC or of tyrosine kinase but not of PKA. Inhibition of PKC or tyrosine kinase itself reduced the expression of collagen I and collagen III mRNA. The mRNA of MMP-2, TIMP-2, and colligin showed the same tendency by stretch. Combined with 10% serum, 6% CMS reduced the mRNA of collagen I (0.62-fold) and collagen III (0.79-fold). Inhibition of PKC or tyrosine kinase, but not of PKA, prevented the reduction of collagen I and collagen III mRNA in 10% serum. The results show that the response of fibroblasts to CMS depends on the serum concentration. At least two signaling pathways are involved in the stretch-induced ECM regulation. Myocardial fibrosis due to ECM remodeling contributes to the dysfunction of the failing heart, which might be attributed to changes in hemodynamic loading.