Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein β-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-β (TGF-β). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-β and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.