Cardiac fibroblasts are responsible for the production of the extracellular matrix of the heart, with alterations of fibroblast function implicated in myocardial infarction and cardiac hypertrophy. Here the role of heterotrimeric GTP-binding proteins (G proteins) in the mechanotransduction of strain in rat cardiac fibroblasts was investigated. Cells in an equibiaxial stretch device were incubated with the photoreactive GTP analog azidoanalido [α-32P]GTP (AAGTP) and were subjected to various regimens of strain. Autoradiographic analysis showed a 42-kDa protein labeled for cells exposed to 12 cycles of 3% strain or 6 cycles of 6% strain over 60 s (strain rate of 1.2%/s), whereas 6 cycles of 3% strain (0.6%/s) elicited no measurable response. To further investigate the role of strain rate, a single 6% cycle over 10 or 60 s (1.2% and 0.2%/s, respectively) was applied, with the more rapid cycle stimulating AAGTP binding, whereas the lower strain rate showed no response. In cells subjected to a single 6% cycle/10 s, immunoprecipitation identified the AAGTP-labeled 42-kDa band as the G protein subunits Gαq and Gαi1. These results demonstrate that G protein activation represents one of the early mechanotransduction events in cardiac fibroblasts subjected to mechanical strain, with the rate at which the strain is applied modulating this response.