Cell-based functional assays are increasingly being utilized for ion channels and other targets in drug discovery. However, development of functional assays is often hampered by problems related to stable expression of ion channels in host cell lines, such as variability in channel activity, cell line degeneration, toxicity associated with gene expression, and time and expense of maintaining the cells in culture. In a previous study, we showed that constitutive expression of the transient receptor potential ankyrin-1 (TRPA1) channel led to cellular toxicity and cell line degeneration. This problem could be circumvented by utilizing large-scale transiently transfected (LSTT) cells, which could be prepared in large quantity and kept frozen at -80 degrees C until needed. LSTT cells from a single preparation were successfully applied toward development of a Ca(2+) influx assay for TRPA1 and a high throughput screening of a >700,000 compound library. In the current study, we extended the application of LSTT cells to Ca(2+) influx assays for transient receptor potential vanilloid-1 (TRPV1), transient receptor potential melastatin-8, and transient receptor potential vanilloid-4 channels. In addition, we found that cryopreserved LSTT cells expressing TRPV1 exhibited the same pharmacology as a TRPV1 stable cell line in the Ca(2+) influx assay. Moreover, by using LSTT cells expressing TRPA1, we successfully developed a membrane potential assay, which gave comparable results to the Ca(2+) influx assay. Hence, the utilization of LSTT cells could reduce the need for stable cell lines, and enable development of functional assays in a more timely and economic fashion for different ion channels and different assay formats.