Low-power laser irradiation (LPLI) can stimulate cell proliferation through a wide network of signals. Akt is an important protein kinase in modulating cell proliferation. In this study, using real-time single-cell analysis, we investigated the activity of Akt and its effects on cell proliferation induced by LPLI in African green monkey SV40-transformed kidney fibroblast cells (COS-7). We utilized a recombinant fluorescence resonance energy transfer (FRET) Akt probe (BKAR) to dynamically detect the activation of Akt after LPLI treatment. Our results show that LPLI induced a gradual and continuous activation of Akt. Moreover, the activation of Akt can be completely abolished by wortmannin, a specific inhibitor of PI3K, suggesting that the activation of Akt caused by LPLI is a PI3K-dependent event. Src family is involved in Akt activation as demonstrated by the part inhibition of Akt activity in samples treated with PP1 (an inhibitor of Src family). In contrast, loading Gö 6983, a PKC inhibitor, did not affect this response. Further experiments performed using GFP-Akt fluorescence imaging and Western blot analysis demonstrate that, the activation of Akt is a multi-step process in response to LPLI, involving membrane recruitment, phosphorylation, and membrane detachment. LPLI promotes cell proliferation through PI3K/Akt activation since the cell viability was significantly inhibited by PI3K inhibitor. All these studies create a concernful conclusion that PI3K/Akt signaling pathway is well involved in LPLI triggered cell proliferation that acts as a time- and dose-dependent manner. J. Cell. Physiol. 219: 553-562, 2009. (c) 2009 Wiley-Liss, Inc.