Extracellular signal-regulated kinase-1 and -2 are both essential for the shear stress-induced human osteoblast proliferation. Extracellular signal-regulated kinases (Erk)-1 and -2 are key mediators of various mitogenic signaling pathways, including mechanical stress-induced osteoblast proliferation. Mechanical stimuli, such as flow shear stress, simultaneously activate both Erk-1 and -2 in osteoblasts, resulting in stimulation of osteoblast proliferation. This study sought to test whether Erk-1, -2, or both are essential for the fluid flow shear stress-induced osteoblast proliferation. Moloney leukemia virus (MLV)-based vectors expressing wild-type (wt)- or kinase-deficient (kd) Erk-1 and Erk-2, respectively, were constructed and used to transduce human TE85 osteosarcoma cells with an MOI of 30. An MLV-red fluorescent protein (RFP) vector was included as a control. Effects of Erk-1 and -2 overexpression on cell proliferation in response to a 30-min constant fluid flow shear stress at 20 dynes/cm2 were determined with [3H]thymidine incorporation 24 h after the shear stress. The MLV-Erk vector-transduced TE85 cells showed a >10- and approximately 2-fold overexpression of Erk-1 and -2 protein, respectively. The RFP expressing control cells and the parental TE85 cells each showed an approximately twofold increase (P < 0.01) in [3H]thymidine incorporation in response to the shear stress. Cells overexpressing wt-Erk-1 or -2 showed small enhancing effects on the response to the shear stress in the increases in [3H]thymidine incorporation and cell number. Conversely, overexpression of kd-Erk-1 or -2 each alone completely abolished the shear stress-induced osteoblast proliferation. Overexpression of either kd-Erk-1 or kd-Erk-2 alone did not have a significant effect on basal osteoblast proliferation, suggesting that the Erk signaling pathway may not be essential for basal cell proliferation. In summary, this study demonstrates for the first time that Erk-1 and -2 are both required for the mitogenic response to fluid flow shear stress in human osteoblasts and that blocking Erk-1 or -2 each alone is sufficient to completely block the mitogenic response to shear stress-induced proliferation.