Extracorporeal shock waves (ESW) have recently been used in bone repair. Extracellular signal-regulated kinase (ERK) and p38 kinase are found to act as important mediators for osteogenic factor and mechanical-stimulated proliferation and differentiation of bone-forming cells. A previous study reported that ESW promoted healing of segmental defects in rats by inducing bone morphogenetic proteins (Bone 32 (2003) 387-396) and stimulating osteogenic differentiation of mesenchymal stem cells. In this study, we found that ERK and p38 activation was involved in ESW-augmented bone regeneration of segmental defects. ESW treatment (0.16 mJ/mm2, 1 Hz, 500 impulses) rapidly promoted [3H]-thymidine uptake in 1 day and progressively increased alkaline phosphatase activity, collagen I, II, and osteocalcin synthesis in callus organ culture within 14 days after treatment. Results of [gamma-32P]-phosphotransferase activity assay showed that ERK and p38 in calluses were rapidly activated 1 day and 7 days after ESW treatment, respectively. Histological observation showed that segmental defects subjected to ESW treatment underwent typical bone formation (mesenchymal cell aggregation, hypertrophic cartilage, and endochondral/intramembrane ossification). Intensive bone formation coincided with evident expression of phosphorylated ERK and p38. Moreover, expression of phosphorylated ERK persisted in mesenchymal, chondral, and osteoblastic cells at newly developed bone and cartilage, and the expression of activated p38 was evident on chondral cells located at hypertrophic cartilage. Our findings suggest that mitogen-activated protein kinases (MAPK) regulate the stimulation of biophysical ESW, triggering mitogenic and osteogenic responses in the defects. ERK phosphorylation is active throughout the period of ESW-induced bone regeneration. p38 activation most likely plays an important role in signaling cartilage formation in callus.