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The purpose of this study was to clarify whether p38 MAPK is involved in the process of low intensity pulsed ultrasound (LIPUS) induced osteogenic differentiation of human periodontal ligament cells (HPDLCs).HPDLCs were isolated from premolars extracted for orthodontic reasons from healthy adolescences and used in the study at passage 5. They were pretreated with p38 specific inhibitor SB203580 and exposed daily to LIPUS with frequency of 1 MHz and intensity of 90 mW/cm2. Osteogenic differentiation was assayed by levels of alkaline phosphatase (ALP) and osteocalcin as well as calcium deposition. The levels of phosphorylated p38 (p-p38) and total p38 in HPDLCs in response to LIPUS for different times were detected by Western blot.The enhanced ALP levels in media and cell lysate, osteocalcin level in media, as well as number of calcium nodules after LIPUS stimulation were decreased by SB203580 treatment. LIPUS stimulation did not change total p38 level, but time-dependently enhanced the level of p-p38; such enhancement was significantly blocked by preincubation with 10 μmol/L SB203580.The p38 MAPK is involved in the process of LIPUS-induced osteogenic differentiation of HPDLCs.