The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BDtrade mark Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.