An apparatus was developed to apply positive or negative hydrostatic pressure dynamically to periodontal ligament (PDL) cells in vitro. The objective of this investigation was to construct this apparatus and to determine its effects on PDL cells. Human PDL cells were collected from freshly extracted premolars. At the sixth passage, the cells were mechanically stimulated by this apparatus at different magnitudes of continuous positive or negative hydrostatic pressures (PHP or NHP, respectively). The application of PHP between 0.3 and 30 gm/cm2 significantly enhanced prostaglandin E (PGE) production and intracellular cyclic AMP (cAMP) of the cells. In contrast, perturbation by NHP significantly decreased PGE production and intracellular level of cAMP. Proliferation rate increased significantly at 24 and 48 hours due to stimulation of these cells with -30 gm/cm2 of NHP. Challenging these cells with +30 gm/cm2 of PHP significantly decreased the proliferation rate of these cells at 24 and 48 hours. Stimulation by PHP between +30 to +600 gm/cm2 increased cell length and width and appeared to increase surface area attachment to the bottom of the culture dishes. In contrast, NHP (between -30 and -600 gm/cm2) decreased these dimensions and appeared to reduce the surface area of attachment. These results indicate that this type of mechanical perturbation of PDL cells produces physiologic responses and is not detrimental to their vitality.