For decades, low-level laser therapy (LLLT) has widespread applications in tendon-related injuries. Although the therapeutic effect of LLLT could be explained by photostimulation of target tissue and cells, how tenocytes sense photonic energy and convert them into cascades of cellular and molecular events is still not well understood. This study was designed to elucidate the effects of LLLT on cell proliferation and collagen synthesis by examining the associated second messengers including ATP, Ca(2+), and nitric oxide using rat Achilles tenocytes. Moreover, proliferating cell nuclear antigen (PCNA) and transforming growth factor-β1 (TGF-β1) related to cell proliferation and matrix metabolism were also studied. The results showed that 904 nm GaAs laser of 1 J/cm(2) could significantly increase the MTT activity and collagen synthesis of tenocytes. Second messengers including ATP and intracellular Ca2+ were increased after laser treatment. Quantitative PCR analysis of tenocytes treated with laser revealed up-regulated expression of PCNA, type I collagen, and TGF-β1. Besides, laser-induced TGF-β1 expression was significantly inhibited by extracellular signal-regulated kinase (ERK) specific inhibitor (PD98059). The findings suggested that LLLT stimulated ATP production and increased intracellular calcium concentration. Directly or indirectly via production of TGF-β1, these second messengers mediated the proliferation of tenocytes and synthesis of collagen.