Utilizing an in vivo model of trabecular bone formation, we demonstrated the temporal and spatial activation of pp125FAK in response to specific mechanical load stimuli. Bone chambers equipped with hydraulic actuators were aseptically inserted into each proximal tibial metaphysis of adult, male dogs under general anesthesia. The load stimulus consisted of a trapezoidal waveform, with a maximum compressive load of 17.8 N, loading rate of 89 N/s, at 1 Hz frequency. One chamber was loaded for 2 (120 cycles), 15 (900 cycles), or 30 min (1,800 cycles), whereas the contralateral chamber served as unloaded control. Bone chambers were biopsied at postload time points of 0, 15, and 45 min. Load-induced activation of FAK was rapid, and the duration of activation was dependent on the number of applied load cycles. Mechanical stimulation increased the association of FAK with Src and the time course of complex formation paralleled the temporal activation of FAK. Evaluation of cryosections revealed prominent FAK immunoreactivity among marrow fibroblasts and stromal cells.