Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca²⁺]_i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca²⁺]_i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca²⁺]_i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1–34) [bPTH(1–34)] significantly enhanced the shear magnitude-dependent increase in [Ca²⁺]_i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca²⁺]_i response to shear and shear + bPTH(1–34). Nifedipine (Nif), an L-type voltage-sensitive Ca²⁺ channel (VSCC) blocker, also significantly reduced the [Ca²⁺]_i response to shear + bPTH(1–34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca²⁺ response to shear, whereas PKA activation significantly increased the [Ca²⁺]_i response to lower magnitudes of shear. bPTH(1–34), which activates both pathways, induced the greatest [Ca²⁺]_i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca²⁺]_i response to shear primarily via PKA modulation of the MSCC and VSCC.