Intracellular Ca(2+) spikes trigger cell proliferation, differentiation and cytoskeletal reorganization. In addition to Ca(2+) spiking that can be initiated by a ligand binding to its receptor, exposure to electromagnetic stimuli has also been shown to alter Ca(2+) dynamics. Using neuronal cells differentiated from a mouse embryonic stem cell line and a custom-built, frequency-tunable applicator, we examined in real time the altered Ca(2+) dynamics and observed increases in the cytosolic Ca(2+) in response to nonthermal radiofrequency (RF)-radiation exposure of cells from 700 to 1100 MHz. While about 60% of control cells (not exposed to RF radiation) were observed to exhibit about five spontaneous Ca(2+) spikes per cell in 60 min, exposure of cells to an 800 MHz, 0.5 W/kg RF radiation, for example, significantly increased the number of Ca(2+) spikes to 15.7+/-0.8 (P<0.05). The increase in the Ca(2+) spiking activities was dependent on the frequency but not on the SAR between 0.5 to 5 W/kg. Using pharmacological agents, it was found that both the N-type Ca(2+) channels and phospholipase C enzymes appear to be involved in mediating increased Ca(2+) spiking. Interestingly, microfilament disruption also prevented the Ca(2+) spikes. Regulation of Ca(2+) dynamics by external physical stimulation such as RF radiation may provide a noninvasive and useful tool for modulating the Ca(2+)-dependent cellular and molecular activities of cells seeded in a 3D environment for which only a few techniques are currently available to influence the cells.