Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca²⁺ signaling and ATP-induced Ca²⁺ oscillations, both of which are modulated by PKC. Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity. Mechanical stimulation of a single cell increased the cytosolic Ca²⁺ concentration in 35-55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (sµG). In outgrowth cultures, 12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca²⁺ "waves" to about 10 cells in unit G and to significantly fewer cells in sµG. TPA only slightly reduced the spread of Ca²⁺ waves in epithelial sheets kept in sµG but did not inhibit Ca²⁺ waves of sheets kept in unit G. In both cell preparations from both conditions, TPA inhibited ATP-induced Ca²⁺ oscillations; however, the effect was more pronounced in cells kept in sµG. These results suggest that PKC activation is more robust in cells subjected to sµG.