Fibroblasts in mechanically unloaded collagen matrices had low levels of DNA synthesis compared with cells in mechanically loaded matrices. Under the former conditions, the cellular ERK signaling pathway appeared to be disrupted. Also, pharmacologic inhibition of ERK signaling blocked DNA synthesis by fibroblasts in mechanically loaded matrices. These results were consistent with the idea that mechanoregulation of fibroblast DNA synthesis in collagen matrices occurs at the level of the ERK signaling pathway.