Helper T cell subsets have evolved to respond to different pathogens, and upon activation secrete distinct sets of cytokines. The discovery and identification of Th17 cells, which develop via a unique lineage from Th1 and Th2 cells, have provided new insights into aspects of immune regulation and host defense that were previously unclear. A key early signaling event upon Ag recognition is elevation of intracellular free Ca2+, and cytokine expression can be differentially induced depending on the duration, amplitude, and pattern of Ca2+ signaling. Th1 and Th2 cells can be distinguished by their Ca2+ profiles, and we provide in this study the first report regarding Ca2+ signaling in Th17 cells. Th17 cells have a distinct Ca2+ signaling profile from Th1 and Th2 cells with intermediate sustained Ca2+ levels and increased oscillations compared with Th2 cells. Elevated intracellular Ca2+ has been shown to inhibit T cell motility, and we observed that Th17 cells, like Th1 cells, are less motile than Th2 cells. Analysis of NF-AT nuclear localization revealed that Th1 and Th17 cells have significantly higher levels at later time points compared with Th2 cells. Thus, these findings show that Th17 cells, in addition to their distinct cytokine response from Th1 and Th2 cells, display unique patterns of intracellular Ca2+ signaling and Th1-like motility behavior and nuclear localization of NF-AT.