A modification of a competitive protein-binding method for the determination of inositol (1,4,5)-trisphosphate ( (\left.\operatorname{Ins}(1,4,5) P_{3}\right)), facilitated by the use of a cell harvester, is described. The method is based on the competition of (\left[{ }^{3} H\right] I n s(1,4,5) P_{3}) with (\operatorname{Ins}(1,4,5) P_{3}) in the sample for binding to a binding protein prepared from bovine adrenal cortex. The assay is carried out in 96well microtiter plates in a final volume of (100 \mu \mathrm{l}) and free (\left[{ }^{3} \mathrm{H}\right] \mathrm{Ins}(1,4,5) \mathrm{P}{3}) is separated from bound by filtration using a cell harvester. This allows the rapid measurement of large numbers of samples, with high reproducibility. Ins ((1,4,5) P{3}) bound to a single class of high-affinity receptors with a (K_{D}) of (2.3 \pm 0.2 \mathrm{~nm}) and a (B_{\max }) of (289 \pm \mathbf{7 ~ f m o l} / \mathbf{m g}) of protein. The binding was rapid, reaching equilibrium after (20 \mathrm{~min}), and reversible. The sensitivity of the assay allows the determination of amounts of (\operatorname{Ins}(1,4,5) P_{s}) as small as (0.1 \mathrm{pmol}), which corresponds to a concentration of (5 \mathrm{nM}) in the sample. The application of the method to measure the formation of (\operatorname{Ins}(1,4,5) P_{3}) in DDT ({1}) MF-2 smooth muscle cells activated with ATP or bradykinin is described. The modification is a rapid, sensitive, convenient, and relatively inexpensive method for the determination of Ins ((1,4,5) P{3}) in large numbers of samples. 1983 Academic Pres, Inc.