The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.